All of the non-structural proteins of poliovirus, including their processing precursors, are involved in the replication of the viral RNA genome. These proteins assemble into a replication complex, which also contains the viral RNA and cellular factors. An understanding of how these viral proteins interact with each other would enhance our understanding of the molecular events occurring during poliovirus infection of the cell. Previously, we have employed the yeast two-hybrid system to construct two separate linkage maps for the polioviral P2 and P3 proteins, respectively. In the present study, we have searched for interacting pairs between the P2 and P3 proteins in a similar inducible yeast two-hybrid system. Although, the primary functions of the proteolytic products of the P2 and P3 domains of the polyprotein in the viral life cycle are different, we observed significant interactions between 2C(ATPase) and 3AB; 2A(pro) and 3A, 3C(pro) or 3D(pol); 2B and 3A or 3AB. All of the interactions were measured in the yeast two-hybrid system by exchanging the interacting pairs on the transcription-activation and DNA-binding constructs. In vitro GST pull-down assay suggested that the 2C(ATPase)/3AB interaction involves both ionic and hydrophobic contacts between the two proteins. The possible biological implication of the interactions observed in the yeast two-hybrid system will be discussed.