Acycloguanosine [9-(2-hydroxyethoxymethyl)guanine; acyclo-Guo] is a potent inhibitor of herpes simplex viruses (HSV); it is selectively phosphorylated in virus-infected cells. In order to define those viral functions that may mediate resistance to acyclo-Guo, the drug sensitivities of temperature-sensitive (ts) and phosphonoacetic acetic acid (PAA)-resistant mutants of HSV-1 and HSV-2 have been determined. Two distinct viral genetic loci are independently associated with acyclo-Guo resistance. Mutations resulting in diminished thymidine kinase activity are associated with resistance to inhibition by acyclo-Guo. Several PAA-resistant viruses that express wild-type levels of thymidine kinase activity are also resistant to acyclo-Guo. This suggests the importance of the viral DNA polymerase region in mediating acyclo-Guo resistance and is consistent with a close relationship between the PAAr mutation site and the AGGr locus. When wild-type HSV-1 is serially propagated under the selective pressure of acyclo-Guo, rapid emergence of resistant virus occurs, accompanied by the simultaneous appearance of thymidine kinase-deficient progeny.