The post-translational regulation of picornavirus gene expression mediated by the cascade processing of viral proteins is not well understood. Both pulse-chase studies of infected cells and in vitro studies of the translation of poliovirus type 1 RNA transcribed from genomic cDNA clones indicate a specific cascade of polyprotein processing in which the P1, P2, and P3 precursor proteins are primary products of viral proteinase cleavage. We report the results of a short-time kinetic analysis of poliovirus type 1 protein processing in an in vitro translation system and in infected HeLa cells which indicate the existence of another, rapid pathway of polyprotein processing mediated by the activity of the 3C proteinase. The observed pathway is distinct from and in addition to the one previously known. The potential role of this alternative pathway of processing in the post-translational regulation of viral gene expression is discussed.