§ Bob Hoskins plays the role of Edward "Eddie" Valiant; this would go on to become one of his most notable roles.
§ Bob Hoskins as Edward "Eddie" Valiant, an alcoholic private investigator who holds a grudge against Toons. Producer Steven Spielberg's first choice for Eddie Valiant was Harrison Ford, but Ford's price was too high. Bill Murray was also considered for the role; however, due to his method of receiving offers for roles he missed out.
§ Charles Fleischer provides the voice of Roger Rabbit, an A-list Toon working for Maroon Cartoons. Roger is framed for the murder of Marvin Acme, and requests Eddie's help in proving his innocence. To facilitate Hoskins' performance, Fleischer dressed in a Roger bunny suit and "stood in" behind camera for most scenes. Animation director Richard Williams explained Roger Rabbit was a combination of "Tex Avery's cashew nut-shaped head, the swatch of red hair...like Droopy's, Goofy's overalls, Porky Pig's bow tie, Mickey Mouse's gloves and Bugs Bunny like cheeks and ears." Fleischer also provides the voices of Benny the Cab and two members of Doom's Weasel Gang, Psycho and Greasy. Lou Hirsch, who supplied the voice for Baby Herman, was the original choice for Benny the Cab, but was replaced by Fleischer.
§ Christopher Lloyd as Judge Doom, the extremely cold-hearted and power-hungry judge of Toontown District Superior Court. Lloyd was cast because he previously worked with Zemeckis and Amblin Entertainment in Back to the Future. Lloyd compared his part as Doom to his previous role as the Klingon commander Kruge in Star Trek III: The Search for Spock, both being overly evil characters which he considered being "fun to play". Lloyd avoided blinking his eyes while on camera in order to perfectly portray the character.
§ Kathleen Turner provides the uncredited voice of Jessica Rabbit, Roger Rabbit's stunningly beautiful and flirtatious Toon wife. She loves Roger because, as she says, "he makes me laugh." Amy Irving supplied the singing voice, while Betsy Brantley served as the stand-in.
§ Joanna Cassidy as Dolores, Eddie's on-off girlfriend who works as a waitress.
§ Alan Tilvern as R. K. Maroon, the short-tempered and manipulative owner of "Maroon Cartoon" studios. This was Tilvern's final theatrical performance.
§ Stubby Kaye as Marvin Acme, prankster-like owner of the Acme Corporation.
§ Lou Hirsch provides the voice of Baby Herman, Roger's middle-aged, foul-mouthed, cigar-chomping co-star in Maroon Cartoons. Williams said Baby Herman was a mixture of "Elmer Fudd and Tweety crashed together". April Winchell provides the voice of Mrs. Herman and the "baby noises".
§ David Lander provides the voice of Smart Ass, the leader of the weasels.
§ Richard LeParmentier has a small role as Lt. Santino. Joel Silver makes a cameo appearance as Raoul, a director frustrated with Roger Rabbit's antics. Frank Sinatra performed "Witchcraft" for the animated Singing Sword. In addition to David Lander as Smart Ass and Charles Fleischer as Greasy and Psycho, Fred Newman voiced Stupid and June Foray voiced Wheezy. Foray also voiced Lena Hyena, a hag Toon woman who resembles Jessica Rabbit and provides a comical role which shows her falling for Eddie and pursuing him. Mel Blanc voiced Bugs Bunny, Daffy Duck, Tweety Bird, Porky Pig and Sylvester (Who Framed Roger Rabbit was one of the final productions in which Blanc voiced these characters before his death in 1989). Animation director Richard Williams voiced Droopy. Joe Alaskey voiced Yosemite Sam and Foghorn Leghorn. Wayne Allwine voiced Mickey Mouse.