A defined in vitro DNA packaging system of phage T3, which is composed of purified proheads and two packaging proteins, the products of genes 18 and 19 (gp18 and gp19, respectively), displayed a DNA-dependent ATPase activity. ATP was hydrolyzed to ADP and Pi. The ATPase activity was stimulated by nonpackageable DNA, such as single-stranded or circular DNA, or RNA (nonpac-ATPase). Among the inhibitors of DNA packaging, actinomycin D specifically inhibited the ATPase activity that was tightly coupled to DNA packaging (pac-ATPase), but did not inhibit the nonpac-ATPase activity. Both activities depended upon a functional packaging complex, but the nonpac-ATPase, once activated, did not require DNA. Unpackageable pUC18 DNA inhibited the pac-ATPase and the phage yield in parallel. Approximately one molecule of ATP was hydrolyzed during the translocation of 1.8 bp of T3 DNA.