Papovaviruses are valuable models for the study of DNA replication in higher eukaryotic organisms, as they depend on host factors for replication of their DNA. In this study we investigate the interactions between the human papillomavirus type 11 (HPV-11) origin recognition and initiator protein E1 and human polymerase α/primase (pol α/primase) subunits. By using a variety of physical assays, we show that both 180- (p180) and 70-kDa (p70) subunits of pol α/primase interact with HPV-11 E1. The interactions of E1 with p180 and p70 are functionally different in cell-free replication of an HPV-11 origin-containing plasmid. Exogenously added p180 inhibits both E2-dependent and E2-independent cell-free replication of HPV-11, whereas p70 inhibits E2- dependent but stimulates E2-independent replication. Our experiments indicate that p70 does not physically interact with E2 and suggest that it may compete with E2 for binding to E1. A model of how E2 and p70 sequentially interact with E1 during initiation of viral DNA replication is proposed.