The replication of bovine papilloma virus (BPV) DNA in vivo requires two viral-encoded proteins, E1 and E2, while all other proteins are derived from the host. We described previously the isolation of the E1 protein and showed that it contains multiple functions required for BPV DNA replication. The BPV transcription factor E2 was shown by others to stimulate BPV DNA replication in vitro. Here, we present results that account for the role of the E2 protein. The E1 protein bound selectively to the BPV minimal origin of replication. This process required MgCl2 and ATP for maximal efficiency. The E1 protein also catalyzed a BPV origin-dependent DNA unwinding reaction. In this report, we show that at low levels of E1 protein, origin binding could be stimulated up to 40-fold by the E2 protein, provided that the DNA contained an E2 binding site. Consistent with this result, the E2 protein stimulated the origin-specific unwinding reaction catalyzed by E1, but it had no effect on the nonspecific E1-catalyzed helicase activity. In the absence of an E2 binding site, both origin-dependent binding and unwinding reactions with the E1 protein were unaffected by the E2 protein. These results suggest that E2 participates in the initiation of BPV DNA replication by enhancing El binding to the BPV origin via DNA-protein and protein-protein interactions.