Purified recombinant poliovirus polypeptide 3AB interacts with 3CDpro and 3Dpol as shown by coimmunoprecipitation with anti-3Dpol antibodies. A consequence of this interaction is an accelerated autoprocessing of 3CDpro to produce 3Cpro and 3Dpol. The activation of 3Dpol polymerase activity by cleavage of 3CDpro, a polypeptide that has no polymerase activity, can be shown by template- and primer-dependent poly(U) synthesis. Anti-VPg antibodies (VPg = 3B) added to HeLa translation extracts programmed with poliovirion RNA inhibit cleavage of 3CDpro whereas addition of purified 3AB or VPg to these translation reactions increases 3CDpro processing. 3AB stimulates also 3Cpro-related proteolysis of 2BC, a poliovirus-specific, nonstructural processing intermediate. In contrast, 3CDpro-specific cleavage of the structural precursor P1 is inhibited by the addition of 3AB as shown by a decrease in the production of VP0 and VP3. These data shed new light on a phenomenon in the regulation of expression of poliovirus genetic information: whereas the proteinase 3CDpro is needed for processing of the capsid precursor, the cleavage product of this relatively stable precursor is required for RNA replication.