The E1 and E2 proteins encoded by papillomaviruses are required for viral replication. Earlier studies have shown that the viral E2 protein plays an important role in replication by targeting the E1 helicase to the origin of replication (ori). We have previously shown that the E1 protein of human papillomavirus (HPV) type 1 is sufficient for the in vivo replication of ori plasmids, although the E2 protein stimulates replication. In this study, we have further analyzed the role of the E2 protein in HPV-1 replication. The optimal ori of HPV-1 contains one putative E1 binding site (E1BS) and two putative E2 binding sites, E2BS-3 and E2BS-4. Plasmid pori171, containing the optimal ori, replicates to much higher levels than plasmid pori312, which includes an additional upstream E2 binding site, E2BS-2, located 75 nucleotides upstream of E2BS-3. To study the possible role of E2BS-2 and other upstream sequences in E2-dependent downregulation of replication, transient replication analysis was done in the presence of increasing levels of the E2 protein. Interestingly, inhibition of pori312 replication was more severe at higher levels of E2, suggesting that this protein may also negatively regulate HPV-1 replication. Deletion of sequences from pori312 containing an additional putative E2BS, E2BS-2A, relieved the repression of replication to a significant extent, while replacement of E2BS-2 with a different sequence of the same length had a modest effect. These results suggest that E2BS-2A plays a major and E2BS-2 a minor role in the negative regulation of HPV-1 replication at high E2 levels. Electrophoretic mobility-shift assays showed that the purified E2 protein bound with high affinity to E2BS-3 and weakly to the other putative E2BSs located within the viral long control region. EMSA using various ori fragments showed the formation of multiple E2-DNA complexes which likely represent binding of E2 to multiple E2BSs present within the HPV-1 ori. Our data are consistent with the assembly of ori-protein complexes at high E2 levels that are impaired for replication and further suggest that E2 may regulate HPV-1 replication by a mechanism involving interaction between the E2 protein bound to E2BSs at a distance.