Phosphonoacetate has been found to inhibit specifically the replication of herpes-viruses. A partial inhibition of vaccinia virus represents the only activity outside the herpesvirus class. The drug was found to be a specific inhibitor of the virus-induced DNA polymerases. Normal cellular polymerases were relatively insensitive to phosphonoacetate, resulting in low cellular toxicity. Our working hypothesis is that the drug binds to the enzyme and that initiation of polynucleotide synthesis occurs in the presence of the drug and the required template, substrates, and cations. However, addition of deoxynucleosides to the elongating nascent chain is prevented by the enzyme-bound drug. Kinetic analyses indicated that phosphonoacetate did not interfere with the binding of DNA template to polymerase; and it did not compete with nucleotide substrate binding. The highly specific inhibitory effects of phosphonoacetate allowed for the selection of partially resistant strains of HSV. Resistance of virus to the drug in cell culture was directly correlated with the same relative resistance of the corresponding cell-free DNA polymerases. Phosphonoacetate was also effective therapeutically in herpesvirus skin and ocular infections in animals. Intraperitoneal administration of the drug reduced death and severity of disease in experimental encephalitis in hamsters. High specificity, low toxicity, and reproducible efficacy in lower animals suggested that phosphonoacetate could be a useful new antiviral drug. Sensitivity to phosphonoacetate also is a useful research tool as a genetic marker for herpesviruses.