Phosphonoacetic acid (PAA) inhibited the synthesis of herpes simplex virus DNA in infected cells and the activity of the virus-specific DNA polymerase in vitro. In the presence of concentrations of PAA sufficient to prevent virus growth and virus DNA synthesis, normal amounts of early virus proteins (alpha- and beta-groups) were made, but late virus proteins (gamma-group) were reduced to less than 15% of amounts made in untreated infected cells. This residual PAA-insensitive synthesis of gamma-polypeptides occurred early in the virus growth cycle when rates were identical in PAA-treated and untreated infected cells. Passage of virus in the presence of PAA resulted in selection of mutants resistant to the drug. Stable clones of mutant viruses with a range of drug sensitivities were isolated and the emergence of variants resistant to high concentrations of PAA involved the sequential selection of mutants progressively better adapted to growth in the presence of the drug. Increased drug resistance of virus yield or plaque formation was correlated with increased resistance of virus DNA synthesis, gamma-protein synthesis, and resistance of the virus DNA polymerase reaction in vitro to the inhibitory effects of the drug. PAA-resistant strains of herpes simplex virus type 1 (HSV-1) complemented the growth of sensitive strains of homologous and heterologous types in mixed infections in the presence of the drug. Complementation was markedly dependent upon the proportions of the resistant and sensitive partners participating in the mixed infection. Intratypic (HSV-1A X HSV-1B) recombination of the PAA resistance marker(s), Pr, occurred at high frequency relative to plaque morphology (syn) and bromodeoxyuridine resistance (Br, thymidine kinase-negative phenotype) markers, with the most likely order being syn-Br-Pr. Recombinant viruses were as resistant or sensitive to PAA as the parental viruses, and viruses recombinant for their PAA resistance phenotype were also recombinant for the PAA resistance character of the virus DNA polymerase. The results provide additional evidence that the herpesvirus DNA polymerase is the site of action of PAA and illustrate the potential usefulness of PAA-resistant mutants in genetic studies of herpesviruses.