Clinical isolates of human cytomegalovirus (HCMV) were screened for susceptibility to ganciclovir by plaque-reduction assay and in situ ELISA. A pretreatment isolate of HCMV obtained from the bronchial brushing of a heart transplant recipient contained both ganciclovir-susceptible and -resistant virus. Ganciclovir-susceptible (P8) and -resistant (D16) strains were further isolated by plaque purification. Both strains phosphorylated ganciclovir at levels similar to the ganciclovir-susceptible strain AD169. D16 was also resistant to phosphonoformic acid and to (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and cytosine. These data suggest that the resistance of D16 to these drugs results from a mutation in the viral DNA polymerase gene.