Oct-1, also referred to as NFIII, OTF-1, OBP100 or NF-A1, is a ubiquitous sequence-specific DNA binding protein that activates transcription and adenovirus DNA replication. The protein contains a conserved DNA binding domain (POU domain) present in several transcription factors. We have overproduced oct-1, the related oct-2 and several oct-1 deletion mutants in a vaccinia expression system to identify the domains important for activation of DNA replication in vitro. Both oct-1 and oct-2 stimulate adenovirus DNA replication in an octamer-dependent manner. From deletion studies it appears that the 160 amino acid long POU domain suffices for stimulation. This domain consists of two subdomains, a POU-specific and a homeo domain. Deletion of the POU-specific domain revealed that the homeo domain has an intrinsic, but weak DNA binding activity and surprisingly, inhibits DNA replication. As the POU domain does not coincide with the transcription activation domain, these results indicate that, although oct-1 functions both in DNA replication and transcription, the mechanisms underlying these processes are probably distinct.