Five chromatographically distinct DNA-dependent ATPase activities have been identified in high salt-detergent extracts of the Novikoff hepatoma. One of these, ATPase III, has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis and has a specific activity of 12 mumol of ATP hydrolyzed min-1 (mg of protein)-1. The enzyme, a dimer of Mr 65000 subunits, has a sedimentation coefficient of 7.0 S in both high salt and low salt, a Stokes radius of 43 A, and a frictional coefficient of 1.31. In the presence of Mg2+ ion and a polynucleotide effector, the enzyme catalyzes hydrolysis of ATP or dATP to a diphosphate with a Km of 206 microM and 110 microM, respectively, for the two substrates. Although single-stranded effectors are preferred, the enzyme has significant activity with double-stranded effectors. The Km for effector is 0.4 microM (nucleotide). The analogues adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), dideoxyadenosine triphosphate (ddATP), and adenosine 5'-(alpha, beta-methylenetriphosphate) (alpha, beta-Me-ATP) are competitive inhibitors of the enzyme while adenosine tetraphosphate (ATP-P), 8-bromoadenosine 5'-triphosphate (8-Br-ATP), 5'-adenylyl imidodiphosphate (AMP-PNP), and adenosine 5'-(beta, gamma-methylenetriphosphate) (beta, gamma-Me-ATP) do not inhibit. The enzyme is insensitive to nalidixic acid, novobiocin, and berenil but is sensitive to N-ethylmaleimide. ATPase III is capable of stimulating DNA polymerase beta on duplex DNA, but this effect is abolished in the presence of ATP gamma S. Polymerase stimulation is further enhanced in the presence of a single-stranded DNA-binding protein. These data suggest that ATPase III may play a role in DNA repair.