A cellular protein that binds to the AT-rich late segment of the simian virus 40 (SV40) origin of replication has been identified as transcription factor Oct1. This conclusion is based on the following observations: the late origin binding protein has a molecular mass of about 100 kDa, like factor Oct1, and shares other biochemical properties with Oct1; its binding to the origin is inhibited by antibodies directed against the POU domain of factor Oct1; the isolated POU domain of Oct1 specifically binds to the SV40 late origin region. Thus, the SV40 genome contains binding sites for transcription factor Oct1 in the origin of replication in addition to the previously characterized octamer sites in the viral promoter enhancer. Oct1, bound to the viral origin, negatively affects the DNA unwinding reaction catalyzed by the viral replication initiator T antigen, suggesting that Oct1 may have a role in the regulation of viral replication.