Isolation and characterization of Chinese hamster ovary cell mutants resistant to different DNA polymerase inhibitors (aphidicolin, ara-A and ara-C) have been described. A particular mutant (JK3-1-2A) characterized in detail was found to grow and synthesize DNA in medium containing an amount of aphidicolin tenfold greater than that which completely inhibited the growth and the DNA synthesis of the wild-type cells. An almost twofold increase in the specific activity of the DNA polymerase alpha was seen in this mutant. The mutant DNA polymerase showed altered aphidicolin inhibition kinetics of dCMP incorporation; the apparent Km for dCTP and the apparent Ki for aphidicolin were increased in the mutant. These alterations in the kinetic parameters were, however, abolished upon further purification of the enzyme. Ara-CTP was found to act as a competitive inhibitor of the dCMP incorporation by both the wild type and mutant enzymes. In contrast, the effect of aphidicolin on dCMP incorporation was either competitive (wild-type enzymes) or noncompetitive (mutant enzyme). The data presented showed that the sites of action for aphidicolin and ara-CTP were distinct; likewise the dCTP binding site appeared to be separate from other dNTP(s) binding sites. The drug resistance of the mutant was inherited as a dominant trait.