A new DNA-dependent ATPase named ATPase IV has been purified to apparent homogeneity from Escherichia coli as a by-product of DNA polymerase III purification. The enzyme has a specific activity of 360 mumol of ATP hydrolyzed per min/mg of protein. The purified enzyme exists as monomer with a molecular weight of 81,000. It sediments in a glycerol gradient as a single species of 4.5 S. The enzyme has considerable activity at 0 degree C and has a Q10 of 3.8. In the presence of a DNA effector and magnesium ion, the enzyme will hydrolyze ATP, dATP, GTP, or dGTP to a nucleoside diphosphate plus orthophosphate with a Km of 0.20, 0.50, 0.60, and 1.30 mM, respectively. The guanine nucleotides, however, are only 25-35% as effective as substrates compared with the adenine nucleotides. ATPase IV shows strong substrate inhibition by ATP, but not dATP, above 0.2 mM. The polynucleotide effector requirement can be satisfied by either single-stranded or double-stranded DNA. The enzyme binds the effector very tightly with a Km of 3 X 10(-8) M (nucleotide) for G4 DNA. The enzyme is inhibited by E. coli single-stranded DNA-binding protein, a variety of ATP analogues and N-ethylmaleimide. The relationship of ATPase IV to DNA polymerase III holoenzyme is discussed.