Using high resolution electron microscopic autoradiography and immunocytochemistry with monoclonal antibodies against poliovirus proteins of the P2 genomic region, the location of these proteins in respect to the virus-induced vesicle formation and the viral RNA synthesis was followed during the viral replication cycle. It was found that P2 proteins become rER associated soon after their synthesis. At the site of protein and rER interaction, electron-dense patches appear. Simultaneously, membrane protrusions grow and form vesicles which finally budd off, carrying the patches on their outer surface. As shown by autoradiography, these patches are the site of viral RNA replication and, therefore, they represent the poliovirus replication complex. The vesicles with the replication complex, including replicating and replicated viral RNA, move away from the rER to form a continuously growing vesiculated area in the center of the infected cell, where virus maturation takes place. A likely function of the 2C protein is to attach the replication complex, or some of its components, to the vesicular membranes.