Clinically acquired acyclovir resistance in herpes simplex has usually been associated with a deficiency in viral thymidine kinase, which, in turn, has been linked with attenuated virulence in animal models. Diminished pathogenicity in thymidine kinase-deficient isolates has been partly responsible for controversies about the clinical significance of antiviral resistance. We report on a series of resistant virus isolates from a patient who had severe, progressive esophagitis. These isolates had various thymidine kinase activities, ranging from 2.8% to 130% when compared with the activity of the isolate obtained before treatment; the resistant isolate 615 retained enzyme activity as well as neurovirulence in an encephalitis model. Plaque purification showed a heterogeneous mixture containing at least one acyclovir-resistant, foscarnet-resistant plaque isolate (615.8) fully able to phosphorylate acyclovir. The 3.3-kbp BamHI fragment containing most of the DNA polymerase gene from isolate 615.8 was purified and used to successfully transfer both acyclovir and foscarnet resistance. Acquisition of in-vitro acyclovir resistance was associated with progression of clinical disease, as well as with maintenance of pathogenicity in an animal model and at least one mutation in viral DNA polymerase. Patients with herpes simplex infections that progress during acyclovir therapy should be observed for acquisition of resistance in the setting of antiviral chemotherapy; future studies should also consider the presence of heterogeneous virus populations in such patients.