The effect of ATP on Escherichia coli DNA polymerase I has been investigated as a function of the concentration of substrates and divalent metal ions in the presence of activated DNA as template. At saturating Mg2+ concentration 1.5 mM ATP stimulated 2.5-times the incorporation of [3H]dGTP when only one substrate (dGTP) was present, and had no significant effect in the presence of all four dNTPs, whereas under similar conditions, a saturating concentration of dATP increased the reaction rate only 1.5-times. At optimal Mn2+ concentrations ATP also showed a similarly marked effect only in the case when one substrate (dGTP) was present in the reaction. The optimal concentration of Mn2+ was shifted by ATP to higher concentrations both in the presence of one and of all four substrates. ATP did not influence the apparent Km for dGTP, while V was increased by a factor of about 2.5. The possible presence of dNTP in ATP, as inpurity, was ruled out by isotope dilution analysis. Thus, ATP stimulated the polymerization reaction only under limited conditions, i.e., when one substrate was present in the reaction.