Once or twice, but never again.

I can think of a few:

• Nicole Jaffe had only one voice role: the "CBS Velma" on Scooby-Doo, Where Are You! and The New Scooby-Doo Movies. Same with her successors on ABC, Pat Stevens and Marla Frumkin. Jaffe reprised the role in a couple of direct-to-DVD Scooby films.
• Indira Stefanianna Christopherson and Heather North both voiced Daphne Blake, and no other characters.
• On The Harlem Globetrotters (1970), all the male voice actors except Scatman Crothers (voice of Meadowlark Lemon) appear to have left voice acting after the Globetrotters' crossovers to The New Scooby-Doo Movies. Likewise Nancy Wible, the voice of Granny, though she had worked in a couple of Loopy de Loop shorts a decade earlier.
• Virginia Gregg's only role for Hanna-Barbera was Tara on The Herculoids, both the original series and the revival on Space Stars.
• Barbara Minkus' only voice role was as Ms. Pac-Man.
  
• In the 1992 series of The Addams Family, Carol Channing and Debi Derryberry voiced Grandmama Addams and Wednesday Addams, respectively.

I was going to add Nancy Cartwright (the voice of Gloria on Richie Rich), but then discoverd that she voiced Woody on Popeye & Son.

Also, I read on imdb that Gloria's last name was Patterson. I thought it was Gloria Glad?

There was also Larry Storch as the Joker on two episodes of "The New Scooby-Doo Movies." Typically Larry Storch didn't do voices for Hanna-Barbera; he was instead heard at Warner Bros. Animation (in the late 1960s), DePatie-Freleng, Filmation and I think a few others...

Uh, Carol Channing was also in "Two Stupid Dogs," in the 1993 episode "Red Strikes Back" as the sexy witch that imprisons the two dogs and Red Riding Hood when they come up to the candy house from "Hansel and Gretel."

Okay...
some major overlooked early ones:
From "Top Cat":
Maurice Gosfield as Benny
Allen Jenkins as Officer Dibble

From "The Jetsons:
Not only Penny Singleton as Jane as mentioned by emarldisle (the original thread poster) but George O'Hanlon as husband George ,though he did write several Gazoo-Flintstones, "Curtain Call in Bedrock" and "Seeing Doubles", both of which had the "Sunshine Babies" closing from the final season debut "No Biz Like Show Biz".

Speaking of No Biz like Show Biz, on the "Flintstones"
Bernard Fox as Brian Epstein, Beatles manager at the time, aka Eppy Briantstone.

Phil Luther (Tink) and Arlene Golonka (Debbie) on "Scoo-" I mean, "Speed Buggy".Edit-just make that a repeat of what the first poster said. Shirley Mitchell did voicework as the Cathy character's mom (not a favorite strip of mine, but..anywy.) and I think she was the hen on WB"s 1952 "Easy Peckin's" ("Oh, George, you shouldn't have done it George, you SHOULDN'T have DONE IT", Bob McKimson, he brought to WB some voice actors that other studios, but not WB used, but that's another topic..

David Hubbard was sometimes billed as David Raynr. He appeared in the NBC series "James at 15/16".

Ted Knight was the narrator of the "Super Friends" hourlong story version in its first year and played the Joker and the Penguin in a "Scooby Doo Movie" story with Batman and Robin. Knight originated his roles as the narrator, Joker, and Penguin in the 1960s Filmation version of "Batman". Knight was a regular performer for Filmation cartoons until his fame as Ted Baxter on "The Mary Tyler Moore Show". Knight also in the voice cast of "Inch High, Private Eye" in a guest role.

Jamie Farr also was a guest voice on "Inch High, Private Eye" and was a story writer for "The Amazing Chan and the Chan Clan". Of course, he was Corporal Klinger on "MASH".

Steve Spears played Blitz in "Trollkins" and Jace in the 80s "Space Ghost" segments on "Space Stars" as his Hanna-Barbera work.

Roger Peltz did a great Jim Nabors impression as Deputy Goofer in "The Buford Files" for his only H-B work.

Jack Mercer reprised his "Popeye" voice for H-B's version of "Popeye" for several seasons and wrote some stories. Maurice LaMarche played Popeye in "Popeye and Son" a few years after Mercer's death in 1984.

Bud Abbott played himself in cartoon form for H-B's "Abbott and Costello" cartoons.

Most of the prime-time stars of H-B's live-action tv show adaptations in cartoon form like "Fonz and the Happy Days Gang", "Laverne and Shirley", "Mork and Mindy", "The Gary Coleman Show," and "The Dukes" played their own characters and that was about it for their H-B involvement for the most part. Henry Winkler reprised his Fonz character in the final "Laverne and Shirley in the Army" season. Sorrell Booke played various guest voices on other H-B cartoons. Robin Williams played in a lot of animation roles in films.

Donny Most did play Ralph Malph in "Happy Days Gang" and Mick in "Teen Wolf" for Southern Star Productions (formerly Hanna-Barbera's Australia studio).

John Erwin played stuffy Ronald Radford in Hanna-Barbera's 1979 version of "The Thing"...Erwin performed on several Filmation shows including Reggie on "Archie", Dick Tracy and Smokey Stover in "Archie's TV Funnies" and He-Man/Prince Adam in the 1980s "He-Man and the Masters of the Universe" show and as the voice of Morris the cat in the 9-Lives commercials.

Hans Conried played villainous Dr. Dred on "Drak Pack" as his only H-B role. He was a familiar voice on Jay Ward's cartoons and of course Captain Hook in Disney's "Peter Pan".

David Landsburg played Woody on "The Buford Files" as his only H-B role.

Diane McClendon played space patrol officer Mini in "Casper and The Angels" as her only H-B role.

Vernee Watson played crime-solving Didi in "Captain Caveman and the Teen Angels" and "Laff-A-Lympics" as her only H-B role.

Nancy Wible's role as Granny, Robert DoQui as Pablo, and Eddie "Rochester" Anderson as Bobby Jo Mason in the 1970s "Harlem Globetrotters" series were their only H-B work. Adam Wade as Twiggy Saunders/Gizmo Man in "Super Globetrotters" was also his only H-B role.

Dom DeLuise as Mr. Evictus in "The Roman Holidays" was his only H-B tv series role. I'm not sure if he participated in any H-B theatrical films.

Tom Bosley played Harry Boyle in "Wait Till Your Father Gets Home" and played a guest role in "Yogi's Gang"...his "Happy Days" Howard Cunningham character didn't appear in "Fonz and the Happy Days Gang". Jack Burns as Ralph and David Hayward as Chet from "Father" were their only H-B roles...Hayward played Chet in Season 1 and Lennie Weinrib took over the role in Season 2.

Vincent Price as Vincent Van Ghoul and Arte Johnson as Weird the ghost in "The 13 Ghosts of Scooby Doo."

Jo Anne Worley as Sgt. Blast in "Private Olive Oyl".

Ron Palillo as Sgt. Squealy in "Laverne and Shirley in the Army".

For the "Charlotte's Web" movie: Debbie Reynolds as Charlotte, Agnes Moorhead as the Goose, and Rex Allen as the narrator as their only HB work...as was Lorne Greene in "Heidi's Song".

Neil Patrick Harris as Max in "Capitol Critters" only.

Storch was a guest voice in the animated "Mork and Mindy" series...a co-production of Hanna-Barbera and Ruby-Spears.

Not quite true. Although Dee Dee Skyes is Vernee's most famous animation role, she also had guest roles on The World's Greatest Super Friends and A Pup Named Scooby-Doo.

The Super Friends franchise itself had several one-role voice actors:

• Shannon Farnon and Constance Caulfield (Wonder Woman)
• Olan Soule (Batman)
• Fernando Escandon (El Dorado)
• Ernie Hudson (Cyborg)
• Dick Ryal (Captain Cold)
• Marlene Aragon (Cheetah)
• Ruth Forman (Giganta)