Two distinct loci that confer resistance to acycloguanosine (acyclo-Guo) in herpes simplex virus types 1 have been identified. The first locus is the gene for the virus-specific thymidine kinase (TK). Mutations that decrease TK activity also render the virus resistant to acyclo-Guo, and the level of resistance corresponds to the decrease in TK activity. acyclo-Guo resistance due to defective TK expression is recessive to the wild-type phenotype, acyclo-Guo-sensitive (ACGs). We term this locus ACGr-TK. The second locus is defined by the properties of a mutant, PAAr5, which is resistant to acyclo-Guo and to phosphonoacetic acid (PAA) yet exhibits wild-type TK activity. The acyclo-Guo-resistant locus in PAAr5 is separable from ACGr-TK mutations by recombination. Moreover, PAAr5 and ACGr-TK mutants can complement each other, producing drug-sensitive gene products which result in growth inhibition in the presence of acyclo-Guo. The acyclo-Guo resistance conferred by PAAr5 behaves as though it were codominant with the wild-type phenotype. This second acyclo-Guo-resistance locus is closely linked to the mutation specifying resistance to PAA. Resistance to PAA is thought to result from mutations in the gene for viral DNA polymerase. Thus, the close linkage of the ACGr and PAAr loci suggest that resistance to both drugs is specified by a mutant DNA polymerase. We term this second locus ACGr-PAA.