Interferons (IFNs) have been shown to suppress the growth of both normal and malignant cells. We examined the effect of gene-cloned IFN-alpha and IFN-gamma on the in vitro activities of human, calf, or rat DNA polymerases. IFN-alpha strongly inhibited the reactions of DNA polymerase alpha and beta at apparent Ki values of 1.25 and 0.35 x 10(5) antiviral units/ml, respectively, but inhibited DNA polymerase gamma only slightly. IFN-gamma inhibited the reaction of DNA polymerase alpha more strongly (Ki, 1.2 x 10(4) units/ml) than IFN-alpha, but not that of DNA polymerase beta. On the other hand, neither IFN-alpha nor IFN-gamma inhibited the reactions of DNA polymerase I from Escherichia coli, Klenow fragment, T-4 DNA polymerase, and RNA polymerase. The fact that Ki values for IFN-alpha of DNA polymerase from calf thymus, human leukemic cells, and rat liver were similar suggests the absence of species specificity among animals with regard to the inhibition of DNA polymerases by IFNs. These results indicate that DNA polymerase may be one of the targets of the action of IFNs.