Fifty-four isolates of cytomegalovirus (CMV) from 25 immunocompromised patients with CMV infections were examined in a plaque reduction assay to determine in vitro susceptibilities to both acyclovir and ganciclovir. Isolates were approximately 25-fold more sensitive to ganciclovir than to acyclovir. The mean ± SD ID50 for all isolates to acyclovir was 63.1 ± 30.2 μM, (median, 52.3 μM; range, 16.7-146.4 μM). The mean ID50 for all isolates to ganciclovir was 2.50 ± 1.27 μM, (median, 2.15 μM; range, 0.65-7.11 μM). Exposure to acyclovir or ganciclovir for periods of 2-5 wk did not alter the mean susceptibility of clinical isolates. However, one CMV strain isolated from a renal transplant patient after 14 days of acyclovir therapy displayed the lowest susceptibility of all strains tested (acyclovir 146.4 μM; ganciclovir 7.11 μM) and may represent selection of a resistant virus population.