Phenotypic and genotypic analyses were done on 17 varicella-zoster virus (VZV) isolates recovered from 10 persons with AIDS (mean CD4 cell count, 16.4/mm3) who had chronic VZV lesions. Eleven acyclovir-resistant isolates were recovered from 10 patients after a mean of 20.1 weeks of therapy. Six susceptible isolates were recovered before acyclovir treatment (n = 1), early during therapy (n = 4; mean time, 4.2 weeks), or after discontinuation of acyclovir (n = 1). Acyclovir-resistant VZV isolates were deficient in thymidine kinase (TK) or induced a TK with altered substrate specificity; all isolates were susceptible to foscarnet. Ten of 11 acyclovir-resistant mutants contained tk gene mutations, including single nucleotide substitutions in highly conserved binding sites (n = 2) as well as nucleotide deletions (n = 4) and insertions (n = 4). These findings suggest that multiple, nonuniform mutations within the tk gene are associated with acyclovir-resistant VZV phenotypes.