The promoter of the human adenovirus type 2 IVa(2) gene, which becomes active only during the late phase of infection, is built largely from sequences spanning, and downstream of, the sites of initiation of transcription. These sequences comprise an initiator, an intragenic sequence necessary for efficient transcription from the promoter by RNA polymerase II, and an intragenic binding site for a cellular repressor of IVa(2) transcription. The properties of the latter protein, which is termed IVa(2)-RF, suggested that it might account for the viral DNA synthesis-dependent activation of IVa(2) transcription during the adenoviral productive cycle. Here we report the results of experiments to assess the contributions of DNA template concentration and IVa(2)-RF binding to the activity of the IVa(2) promoter using a transient expression system. When a IVa(2)-EGFP reporter gene was introduced into HeLa cells, in which IVa(2)-RF was identified, no EFGP synthesis could be detected. In contrast, in IVa(2)-RF-containing cells in which the plasmid carrying the chimeric gene replicated, synthesis of both the EGFP protein and the IVa(2)-EGFP mRNA was readily detected. A vector mutation that blocked plasmid replication reduced IVa(2) promoter activity to undetectable levels. In contrast, a IVa(2) promoter substitution that impaired binding of IVa(2)-RF increased IVa(2) promoter activity under all conditions examined. Furthermore, introduction of DNA containing the IV-RF binding site with the chimeric reporter genes resulted in increased transcription from the IVa(2) promoter in the absence of plasmid replication. These properties are consistent with the hypothesis that the relative concentration of the IVa(2) promoter and of the cellular repressor that binds to it governs transcription from this adenoviral promoter.