The show features a hillbilly cat called Punkin' Puss who lives in a house in the woods of the southern US. Punkin' is preoccupied with a hillbilly mouse called Mushmouse who lives there too, and Punkin' frequently tries to shoot him with his rifle. In many cartoons, one of Mushmouse's cousins visits and gives Punkin' Puss a hard time.

Season 2 of Punkin' Puss & Mushmouse premiered on 1965-09-11.