Human herpesvirus 6 (HHV-6) was examined in vitro for its sensitivity to a broad range of nucleoside analogues, including acyclovir (ACV), ganciclovir (GCV), penciclovir (PCV), buciclovir (BCV), brivudin (BVDU), the N7-isomer of 6-deoxyganciclovir (S2242), foscarnet (phosphonoformic acid, PFA), and several acyclic nucleoside phosphonate (ANP) analogues such as (S)-HPMPA, (S)-HPMPC, PMEA and PMEDAP. Antiviral efficacy was monitored microscopically by the inhibitory effect of the compounds on HHV-6-induced cytopathic effect in human T-lymphoblastoid HSB-2 cells. In addition, a newly developed immunofluorescence/flow cytometric assay (FACS) was used to determine HHV-6-specific antigen expression. A close correlation was observed between the antiviral data obtained by the microscopic assay and the flow cytometric assay. Marked antiviral efficacy was noted for S2242, PFA and the ANP analogues (S)-HPMPA, (S)-HPMPC, (S)-cHPMPC, (S)-3-deaza-HPMPA, (S)-3-deaza-cHPMPA, (S)-HPMPG and (R)-HPMPG. Also, PMEA and PMEDAP proved highly active against HHV-6 infection, whereas (S)-FPMPA and (R)-PMPDAP were inactive. ACV was only slightly protective against HHV-6, and no activity was found for GCV, PCV, BCV and BVDU. Overall, the efficacy of the nucleoside analogues against HHV-6 appeared to correlate with their efficacy against human cytomegalovirus (HCMV).