Yosemite Sam is a Looney Tunes star who now teaches at Acme Looniversity. He is the mentor and favorite teacher of Montana Max. On the Looney Tunes shorts, he is commonly depicted as an extremely grouchy gunslinging prospector or cowboy (or something completely different) with a hair-trigger temper and an intense hatred of rabbits, particularly Bugs Bunny. Along with Elmer Fudd, he is the de facto archenemy of Bugs Bunny. He describes himself as "the meanest, roughest, toughest, rip-roarin'-est, he-man stuffest, Edward Everett Horton-est hombre whose ever crossed the Rio Grande and what ever packed a six-shooter!" Sam uses phrases like "ain't" and "ain't none" and pronouncing his "-ing"s like "-in'"s.
Sam is voiced primarily by Jeff Bergman in Tiny Toon Adventures. Joe Alaskey voices him in Gang Busters and Best of Buster Day. Maurice LaMarche voices him in Two-Tone Town. Charlie Adler voices him in The Wide World of Elmyra.
Yosemite Sam briefly appears in the Season 1 premiere The Looney Beginning, during Bugs' song about Acme Looniversity. He is seen in Exploding Cakes Class. Also in the episode, Buster disguises himself as him in order to get the stolen scripts back from Montana Max.
Although he does not appear in The Wheel O' Comedy segment Win, Lose or Kerplowie, but his face is shown before his name without "I" and "E" is shown, as Monty was about to say that his mentor's name is missing "I" and "E".
In Gang Busters, Sam appears as the judge and jury (which only consists of Yosemite Sam clones), whom have Buster on trial (with an obnoxious Plucky as his lawyer) for stealing a slushie machine (which was stolen by Monty and his gang, who framed Buster with the crime). They find Buster guilty, as well as Plucky, and sentence them to reform school.
In The Wide World of Elmyra episode segment, Turtle Hurdle, Yosemite is driving on a newly built highway and spots Tyrone the Turtle trying to get across it. He tries to run him over, but when he misses, he reverses and makes several more unsuccessful attempts to squash him. A disappointed Sam remarks, "Dag-raddit! I missed!" as he drives away.
In the Looniversity Daze episode segment, The Learning Principal, Sam is the teacher of Class Clowning 101. As he tries to teach the class, Buster Bunny plays tricks on him, and when Sam finds out, he sends Buster to the principal's office. After having a talk with Bugs Bunny, who controls the Great and Powerful Principal, Buster continues to play pranks and eventually gets an A+ in class clowning.
Sam appears brifely in the Music Day episode segment, The Horn Blows at Lunchtime, when the Acme Looniversity students are having a food fight over who "cut the cheese." He exclaims, "Great horny toads!" as Sneezer continues to practice his trumpet, while at the same time, eating Limburger cheese. Embarrassed, Sam says, "Uh, pardon."
In the Best of Buster Day episode segment, Compromising Principals, Yosemite Sam is Vice Principal and holds a Student of The Day Contest, in which the student on his or her best behavior will win free Weenie Burgers for the rest of their life. The reason being is because he is under consideration for a new job as Vice Principal at Miss Conduct's Advanced School of Immaculate Manners. Buster and his friends are worried that if Sam gets the job, he will be replaced with someone competent, and they won't be able to get away with anything. Throughout the cartoon, they misbehave behind Sam's back and cost him his promotion.