After serial passage in the presence of increasing concentrations of ganciclovir (GCV) in vitro, a human herpesvirus-6 (HHV-6) mutant exhibiting a decreased sensitivity to the drug was isolated. Analysis of drug susceptibility showed that the IC 50 of this mutant was 24-, 52-and 3-fold higher than that of the wild-type (wt) IC 50 in the case of GCV, cidofovir and foscarnet, respectively. Genotypic analysis showed two single nucleotide changes as compared to the wild-type : an A G substitution of the U69 protein kinase (PK) gene resulted in an M 318 V amino acid substitution and the other change, located in the C-terminal part of the U38 gene, resulted in an A 961 V amino acid substitution within the DNA polymerase. The M 318 V change was located within the consensus sequence DISPMN of the putative catalytic domain VI of the PK. This change was homologous to the M 460 V and M 460 I changes that had been reported previously within the consensus sequence DITPMN of the human cytomegalovirus (HCMV) UL97 PK and associated with the resistance of HCMV to GCV. The M 318 V change was also detected by PCR in HHV-6-infected PBMCs from an AIDS patient who had been treated with GCV for a long period of time and exhibited a clinically GCVresistant HCMV infection. These findings provide strong circumstantial evidence that the M 318 V change of the PK gene is associated with resistance to GCV and raise the question of cross resistance to this drug among different betaherpesviruses.