Initiation of adenovirus DNA replication is strongly enhanced by two cellular transcription factors, NFI and Oct-1, which bind to the auxiliary origin and tether the viral precursor terminal protein-DNA polymerase (pTP.pol) complex to the core origin. NFI acts through a direct contact with the DNA polymerase, but the mode of action of Oct 1 is unknown. Employing glutathione S-transferase-POU pull-down assays and protein affinity chromatography, we have established that the POU domain contacts pTP rather than pol. The POU homeodomain is responsible for this interaction. The protein-protein contacts lead to increased binding of pTP-pol to the core origin, which is caused by a reduced off-rate. The enhanced formation of a pTP.pol.POU complex on the origin correlates with stimulation of replication. Using an immobilized replication system, we have studied the kinetics of dissociation of the Oct-1 POU domain during replication. In contrast to NFI, which dissociates very early in initiation, Oct-1 dissociates only when the binding site is rendered single-stranded upon translocation of the replication fork. Our data indicate that NFI and Oct-1 enhance initiation synergistically by touching different targets in the preinitiation complex and dissociate independently after initiation.