POU domain proteins constitute a family of eukaryotic transcription factors that exert critical functions during development. They contain a conserved 160 amino acids DNA binding domain, the POU domain. Genetic data have demonstrated that some POU domain proteins are essential for the proliferation of specific cell types, suggesting a possible role in DNA replication. In addition, the ubiquitous POU transcription factor Oct-1 or its isolated POU domain enhances adenovirus DNA replication. Here we compared the DNA binding specificities of POU domain proteins from different subclasses. They exhibit overlapping, yet distinct binding site preferences. Furthermore, purified Pit-1, Oct-1, Oct-2, Oct-6, Oct-4 and zebrafish POU[C] could all stimulate adenovirus DNA replication in a reconstituted in vitro system. Thus, activation appears to depend on a property common to most POU domain proteins. Adenovirus DNA replication is also stimulated by the transcription factor NFI/CTF. In contrast to NFI, the POU domain did not enhance binding of precursor terminal protein-DNA polymerase to the origin nor did it stabilize the preinitiation complex. These results suggest that the POU domain acts on a rate limiting step after formation of the preinitiation complex.