At present, there is no effective therapy for human cytomegalovirus (CMV) infections. Although acyclovir inhibits in vitro clinical isolates of CMV, preliminary human trials suggest that acyclovir may not be successful as a single antiviral agent in treating CMV infections. The antiCMV activity of acyclovir in combination with human fibroblast interferon (IFN-beta), phosphonoformic acid (PFA), or trifluorothymidine (TFT) was therefore evaluated. When acyclovir (20 microM) was combined with IFN-beta (25 U/ml), additive antiviral effects were observed for the four clinical CMV isolates studied and the laboratory adapted strain, AD-169. The combination of acyclovir (20 microM) with PFA (25 microM) was synergistic for all four clinical isolates studied, but additive for the AD-169 strain. Similarly, the combination of acyclovir (20 microM) and TFT (0.17 microM) was synergistic for three of four clinical isolates, and additive for one clinical strain and the AD-169 virus. These findings suggest that acyclovir combined with other antiviral agents may be useful in the treatment of CMV disease.