Human herpesvirus 6 (HHV-6) is latent in the majority of the adult population. Due to its ability of causing opportunistic infections, alone or in concert with the other beta-herpesviruses human cytomegalovirus (HCMV) and HHV-7, its importance as a pathogen in immunocompromised patients has increasingly been recognized. We here report the characterization of the main antiviral target, the HHV-6 DNA polymerase, expressed in a eukaryotic in vitro transcription/translation assay. This technique represents a fast and straightforward approach for the study of existing and new inhibitors of HHV-6 DNA polymerase. The present study shows that ganciclovir is less active against HHV-6, as compared to its activity against HCMV, both in cell culture and at the enzymatic (i.e. DNA polymerase) level. Recently, a mutant HHV-6 strain carrying an amino acid substitution in the ganciclovir phosphorylating pU69 kinase has been isolated both from patients and in cell culture. The strain isolated in vitro, however, carried an additional mutation in the viral DNA polymerase. From our experiments presented here, we conclude that the pU69 M(318)V amino acid substitution rather than the A(961)V substitution in HHV-6 DNA polymerase, is responsible for the ganciclovir-resistant phenotype.