Using transient assays, the cis and trans requirements for human papillomavirus type 31b (HPV-31b) replication in transformed and primary keratinocytes have been determined. We demonstrate that expression of both the E1 and E2 open reading frames are necessary and sufficient for replication of a plasmid construct containing a genomic 291-bp fragment in both cell types. The roles of the E1 and E2 proteins in replication were further examined by overexpressing the gene products in a glutathione-S-transferase bacterial expression system. In addition to a full-length E1 protein, a truncated E1 protein (E1*) which consisted of the amino-terminal 268 aa was synthesized. Both E1 and E1* were found to complex efficiently with the E2 protein in vivo and in vitro. In addition, site-specific DNA binding by both the E2 and E1 proteins to HPV-31b origin containing DNA was observed. The E1* protein, however, failed to bind HPV-31b DNA specifically and could not cooperate with E2 for replication in transient assays. DNase I footprinting demonstrated that the E1 protein of HPV-31b bound to an A/T-rich region (nucleotides 7905-24) which shares similarities with the binding site for the bovine papillomavirus type 1 (BPV-1) E1 protein. These studies describe both similarities and differences in the requirements for replication of HPV-31b and BPV-1.