The effects of the adenovirus type 2 (Ad2) structural proteins on Ad DNA synthesis in vitro have been examined. Both of the viral core proteins, polypeptides V and VII were shown to inhibit Ad2 DNA synthesis in vitro; however, only the major core protein, polypeptide VII, inhibited DNA synthesis at a ratio of protein to DNA proportional to the number of polypeptide VII molecules associated with the Ad2 DNA in the mature virion. In addition, fractions containing the precursor to polypeptide VII, pVII, were capable of inhibiting Ad2 DNA replication in vitro to the same extent as polypeptide VII. Purified polypeptide VII bound to double-stranded DNA with no apparent sequence specificity. In addition, polypeptide VII protected Ad2 DNA from digestion with micrococcal nuclease. The binding of polypeptide VII was probably responsible for the inhibition of Ad2 DNA synthesis in vitro by virtue of rendering the DNA inaccessible to viral replication proteins. These results suggest that the core proteins must be removed from the Ad2 genome before the template can function in genome replication and that assembly of pVII on Ad2 DNA can terminate the replication process.