A single-stranded DNA-dependent ATPase activity, consisting of two subunits of 83 kDa (p90) and 68 kDa (p70), was previously purified from HeLa cells (Vishwanatha, J.K. and Baril, E.F. (1990) Biochem 29, 8753-8759). Homology of the two subunits of single-stranded DNA-dependent ATPase with the human Ku protein (Cao et al. (1994) Biochem 33, 8548-8557) and identity of the Ku protein as the human DNA helicase II (Tuteja et al. (1994) EMBO J. 13, 4991-5001) have been reported recently. Using antisera raised against the subunits of the HDH II, we confirm that the Hela single-stranded DNA-dependent ATPase is the HDH II. Similar to the activity reported for Ku protein, ssDNA-dependent ATPase binds to double-stranded DNA and the DNA-protein complex detected by gel mobility shift assay consists of both the ATPase subunits. The p90 subunit is predominantly nuclear and is easily dissociated from chromatin. The p70 is distributed in cytosol and nucleus, and a fraction of the nuclear p70 protein is found to be associated with the nuclear matrix. Both the p90 and p70 subunits of the ATPase are present in G1 and S phase of the cell cycle and are rapidly degraded in the G2/M phase of the cell cycle.