The inhibition in vitro of herpes simplex virus 1 and vaccinia virus DNA polymerases by aphidicolin is primarily noncompetitive with dGTP, dATP, dTTP, DNA, and Mg2+ and competitive with dCTP in analogy with the mode of inhibition of cellular alpha-polymerase. The degree of inhibition of viral or cellular growth in vivo can be quantitatively predicted by the degree of inhibition of the isolated replicative DNA polymerases at the same concentration of aphidicolin in suitable conditions (limiting dCTP concentration). Thus, the only in vivo target for aphidicolin is probably the replicative DNA polymerase, and aphidicolin is a highly specific inhibitor of replicative nuclear DNA synthesis in eucaryotes. This, coupled with the lack of mutagenic effect, represents a valuable property for an anticancer drug. The specificity of inhibition (contrary to the aspecific effect on almost all DNA polymerases by a true competitive inhibitor, such as 1-beta-D-arabinofuranosylcytidine 5'-triphosphate) and the structure of the drug, which does not resemble that of the triphosphates, suggest that aphidicolin must recognize a site common only to the replicative DNA polymerases of eucaryotes and different from the binding site for deoxyribonucleic triphosphates and DNA, which should be similar in reparative and procaryote-type DNA polymerase; the aphidicolin binding site is probably very near to, or even overlaping with, the binding site for dCTP so that the drug mimics a competitive effect with this nucleotide.