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The Tiny Toon Adventures animated television series features an extensive cast of characters. The show's central characters are mostly various forms of anthropomorphic animals, based on Looney Tunes characters from earlier films and shows. In the series, the characters attend a school called Acme Looniversity, set in the cartoon community of Acme Acres.

Buster Bunny (initially voiced by Charlie Adler and later by John Kassir) is a main character of the show along with Babs Bunny. Buster is a young, blue-and-white male rabbit with a red shirt and white gloves. Bugs Bunny is his mentor. In the last episode, It's a Wonderful Tiny Toon Christmas Special, Babs states that Buster is indeed her boyfriend. Adler voiced Buster in the cancelled video game Tiny Toon Adventures: Defenders of the Universe.

Babs Bunny (voiced by Tress MacNeille) is a main character of the show along with Buster Bunny. Babs dislikes being addressed by full name, Barbara Anne Bunny. She is a young, female pink rabbit with a yellow shirt, violet skirt, and violet bows. She can change her attire by spinning around and can also do impressions of celebrities including Mae West, Popeye, and Jessica Rabbit and a few others. In an episode of Animaniacs, she and Buster arrive to a party and Buster affirms in the reception, "Buster and Babs Bunny, no relation" to Bugs Bunny. Her role model is Honey, the girlfriend of the early Looney Tunes character Bosko.

Plucky Duck (voiced by Joe Alaskey at his "normal" age and Nathan Ruegger as a baby) is a young, green male duck in a white tank top. Much like his Looney Tunes counterpart and idol, Daffy Duck, he is portrayed as greedy, selfish and egotistical, often engaging in various schemes with the goal of personal glory. However, Plucky does have moments of heroism and goodwill. Plucky is friends with Hamton J. Pig and Buster Bunny (although he frequently annoys Buster, again much like Daffy does with Bugs). Plucky constantly pines for the love of Shirley McLoon though she has extremely little patience for him. Also like Daffy Duck, Plucky is capable of flying with his wings but very rarely does so.

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