A simple and reproducible method for detection of human herpesvirus 6 (HHV-6) antigens was developed using a dot blot assay in order to assess virus titer and to evaluate the effect of antiviral drugs against HHV-6. The titer of virus stocks obtained by the dot blot assay was the same as that determined by an immunofluorescence assay (IFA). This method was then applied to evaluate the effect of several antiviral drugs against HHV-6, including phosphonoformic acid (PFA), 9-(2-hydroxyethoxymethyl)guanine (ACV), 9-[(1,3- dihydroxy-2-propoxy)methyl]guanine (DHPG) and (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]cytosine [(S)-HPMPC]. The end-point concentrations (EPC, which was determined visually) of DHPG and (S)-HPMPC were approximately 1 μg ml. These drugs were more effective than the others which had EPCs of approximately 16 μg ml each. The EPC values of four drugs were almost similar to EC90 values determined by measuring density of each dot blot. Thus, the EPC values can be utilized to determine the efficacy of these drugs in the inhibition of HHV-6 replication. The block in virus replication was not due to toxic effect of these drugs on cord blood mononuclear cells (CBMCs). These results suggest that a dot blot method which detects HHV-6 antigens can be useful for titrating virus yield and evaluating antiviral drugs against HHV-6 replication.