Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6), and human herpesvirus type 8 (HHV-8) comprise a group of lymphotropic herpesviruses which are responsible for a wide range of diseases, including lymphoproliferative disorders and tumors. We have developed several flow cytometric assay (FACS) systems to evaluate antiviral efficacy against EBV, HHV-6 and HHV-8. Assays using either EBV or HHV-8, members of the gammaherpesvirus subfamily, have shown that while EBV responds well to acyclovir (ACV), HHV-8 was most sensitive to cidofovir (CDV). Since HHV-6 strains are divided into two sub-groups, A and B, we evaluated antiviral efficacy for strains from each group. The group A strain, HHV-6(GS), was inhibited by foscarnet (PFA), CDV and ganciclovir (GCV) in both Sup-T1 and HSB-2 cell lines. HHV-6(Z-29), a representative group B virus, was inhibited by GCV and CDV but not by PFA. Our findings indicate that flow cytometry can be utilized to efficiently evaluate new antiviral agents against lymphotropic herpesviruses and that the results are comparable to those obtained by other methods such as immunofluorescence.