Jones introduced Hubie and Bertie in the short The Aristo-cat, first released on 19 June 1943. The plot of the cartoon would serve as the template for most future Hubie/Bertie outings: A character with some mental illness or degree of naïveté, here, a cat who doesn't know what a mouse looks like, is psychologically tormented by the pair. In this cartoon, they tell the mouse-hungry cat that a bulldog is a mouse, leading to several painful encounters for the cat. Hubie is voiced by Michael Maltese and Bertie by Tedd Pierce; both men were screenwriters for Jones at the time.
Hubie and Bertie as designed by Jones are nearly identical mice with long snouts, large ears, and big, black noses. The two are completely anthropomorphic, walking on their stubby hind legs and using their forelimbs as arms. The characters are primarily distinguished by their color; one is brown with a lighter-colored belly and face, while the other is gray (which mouse is which color changes from film to film). Hubie has a pronounced Brooklyn street-accent. Bertie has large buck teeth, and a habit of responding to Hubie with: "Yeah-yeah, sure-sure!" or snickering "Riot!" if Hubie has just proposed some scheme with great comedic potential.
Beginning with The Aristo-cat, Jones quickly established differing personalities for his mice. Hubie, here in gray, is the thinker. He comes up with the plans, and he is the mouse with the chutzpah to fast-talk anyone into doing almost anything. Bertie, on the other hand, brown in this cartoon, is the doer. He performs the gruntwork to accomplish Hubie's schemes. Hubie makes it clear who is subservient to whom, slapping the simpler Bertie around whenever his natural goofiness interferes with the task at hand.