Chronic cutaneous herpes simplex virus infection is described in a 68-year-old man who was immunocompromised because of chronic lymphocytic leukemia. The herpes infection was not amenable to therapy with acyclovir. Clinical isolates of herpes simplex virus were assessed for viral thymidine kinase activity, which was markedly decreased in two isolates. By the method of viral plaque autoradiography, these isolates were determined to be composed primarily of mutant thymidine kinase-negative herpes simplex virus mixed with occasional standard thymidine kinase-positive herpes simplex virus. Viral plaque autoradiography permitted the quantitation of proportions of thymidine kinase-negative and thymidine kinase-positive herpes simplex virus in the mixed virus populations. The chronic cutaneous infection persisted, unlike other reported infections by thymidine kinase-negative herpes simplex virus.