Acyclovir-resistant mutants arise under nonselective conditions at relatively high frequencies from recently cloned populations of herpes simplex virus (HSV). The vast majority of mutants isolated following selection for acyclovir-resistance are mutated in the thymidine kinase ( tk) gene. These mutants vary considerably in their acyclovir-resistance and exhibit TK and acyclovir-phosphorylating activity reduced to varying degrees, indicating that they are mutated at different sites within the gene. Genetically, these mutants behave as alleles of a single locus and map physically to the HSV- tk gene. Two mutants selected for acyclovir-resistance carry mutations in the viral DNA polymerase ( pol) gene. One, ACG r4, is mutated in both the tk and pol genes, whereas the acyclovir-resistance phenotype of the other mutant, BW r, can be ascribed to a mutation in the pol gene alone. In addition, mutant PAA r5—selected for resistance to the antiHSV drug phosphonoacetic acid (PAA)—is acyclovir-resistant due to a mutation in its pol gene. Phenotypically, the partially purified polymerases of these mutants exhibit altered susceptibility to acyclo-GTP compared with their wildtype counterparts. Genetically, these mutants behave as if linked to the PAA r marker of the pol gene. Thus, by both biochemical and genetic criteria, acyclovir-resistant mutants have been shown to be mutated in the HSV tk and/or pol genes. These criteria, generally applicable to studies of mutants resistant to any antiviral drug, are discussed critically. Our results indicate that, although all acyclovir-resistant mutants can be ascribed to mutations in either the tk or pol gene, these mutants display a variety of phenotypes including varying degrees of acyclovir-resistance and coresistance to other antiHSV drugs. The possible implications of these phenotypes for the clinical use of acyclovir are discussed.