For the past 2 years, a survey network was established for the screening of acyclovir (ACV)-resistant clinical isolates of herpes simplex virus (HSV). Among 889 strains tested for in vitro ACV sensitivity, 14 HSV-1 and 6 HSV-2 were resistant to ACV concentrations exceeding 3 micrograms/ml. These resistant isolates were most often obtained after prolonged ACV treatment of severely immunocompromised patients. For five patients, the emergence of ACV-resistant virus correlated with treatment failure. In particular, a decrease in the in vitro sensitivity to ACV was observed for eight successive HSV-1 isolates from one immunodeficient patient undergoing therapy. All ACV-resistant isolates were studied for their sensitivity to different antiherpetic compounds and showed various cross-sensitive and -resistant patterns. The examination of viral populations by plaque autoradiography procedures frequently revealed their heterogeneity in terms of thymidine kinase (TK) phenotype and allowed the detection of various proportions of TK-positive (TK+), TK-deficient (TKD), or TK-altered (TKA) viruses. Our data underline the importance of monitoring the emergence of drug-resistant virus during the course of antiviral therapy, and the need for the detection and characterization of TK mutants in clinical specimens. The routine examination of drug sensitivity of HSV isolates provides useful information to clinicians for the management of ACV treatment in the hope of preventing ACV-resistant mutants from becoming predominant in mixed viral populations.