Initiation of bovine papillomavirus (BPV) DNA synthesis in vivo and in vitro depends on the interaction of the viral initiator protein E1 with the replication origin (ori+ DNA). The viral E2 protein assists this interaction, resulting in a cooperative assembly of both proteins on the replication origin. Using gel mobility-shift experiments, we demonstrate that in the presence of both E1 and E2 proteins two classes of ori+ DNA complexes were formed: complex 1 (c1) and complex 2 (c2). Formation of c1 depended on both the E1 and E2 proteins and both proteins were contained within c1. The generation of c2 was dependent on the E1 protein and could be enhanced by E2, but the E2 protein was not detected within c2. At high E2/E1 ratios, c1 was the dominant complex formed. Under these conditions, E1-dependent BPV DNA synthesis in vitro was inhibited. At low E2/E1 ratios, the stimulation of c2 was correlated with the stimulation of BPV DNA replication by E2 in vitro. These data suggest that E2 assists E1 in the formation of an intermediate c1 complex, which is replication inactive. The c1 complex is converted in turn to the replication-active c2 complex, which contains E1 but lacks E2. We propose that the ratios of c1 and c2 formed in response to the levels of E1 and E2 protein determine the potential for BPV DNA synthesis in vitro and in vivo and may contribute to copy number regulation of BPV plasmids within the cell.