A cDNA clone encoding the 3CD proteinase (3CDpro) of poliovirus type 2 (Sabin), the precursor to proteinase 3Cpro and RNA polymerase 3Dpol, was expressed in bacteria by using a T7 expression system. Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3Cpro and 3Dpol. Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CDpro T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing. This mutant was purified to 86% homogeneity and used for subsequent proteolytic studies. Purified 3CDproM (M designates the cleavage site mutant 3CDpro T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide. Purified 3CDproM demonstrated the same detergent sensitivity in processing experiments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates. Purified 3CDproM did not have any detectable RNA polymerase activity, whereas 3Dpol, separated from 3CDproM by gel filtration in the last step of purification, did. We conclude that 3CDproM can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate. Moreover, cleavage of 3CD to 3Dpol is needed to activate the 3D RNA polymerase.