To determine the sensitivity of herpes simplex virus isolates to acyclovir and the importance of resistant isolates in hospitalized patients. Design: Retrospective incidence cohort study. Setting: all herpes simplex virus isolates cultured over 1 year from patients followed at a tertiary care center. Patients: Consecutive herpes simplex virus isolates were collected from 207 patients, including immunocomponent patients, patients with malignancy, neonates, bone marrow and organ transplant recipients, and patients seropositive for human immunodeficiency virus. Measurements and Main Results: A rapid nuclei acid hybridization method was used to assess susceptibility to acyclovir. Acyclovir-resistant herpes simplex viruses were recovered from 7 of 148 immunocompromised patients (4.7%) bur from none of 59 immunocompetent hosts. Clinical disease was found in all 7 patients with resistant herpes simplex virus and was more severe in pediatric patients. All resistant isolates were from acyclovir-treated patients and had absent or altered thymidine kinase activity by plaque autoradiography. Conclusion: Herpes simplex virus resistant to acyclovir arises relatively frequently in immunocompromised patients and may cause serious disease. Rapid detection of resistance permits antiviral therapy to be individualized. Antiviral susceptibility testing to monitor viral resistance should be encouraged, especially in tertiary care settings.