DNA replication of human papillomavirus type 18 is dependent on viral proteins E1 and E2 and the subsequent interaction of these proteins with the viral origin of replication. Using a site-directed mutagenesis analysis, we examined the sequence requirement for the DNA replication of the human papillomavirus type 18. We showed that both the E1BS palindrome and E2BS are the major determinants of the HPV replication efficiency. In particular, abolishing E2 binding sites demonstrated that E2BS makes a significant contribution towards HPV-18 DNA replication. Each part of the 18-bp inverted repeat sequence of the E1BS motif showed a clear functional difference between two regions: nt 13-21 (3' half segment) is evidently more important for replication than nt 4-12 (5' half segment). Besides E1BS and E2BS, cis-acting elements such as the poly-A6 track, perhaps the YY1 binding site, and the TATA box sequence within the origin region exhibited some contributions to optimum replication. In addition, inserting an enhancer region to the minimum origin DNA derivatives increased replication approximately 2-fold compared with the wild type levels and showed some compensational effects on loss of the cis-element within the HPV-18 minimum origin, suggesting that an enhancer region is required for efficient replication of the papillomavirus origin. These results suggest that the formation of an E1-E2-ori complex is important for replication, and other sequences near the E1 and E2 binding sites assist E1-E2-ori-mediated DNA replication in vivo.