Several 2-alkylthio- and 2-benzylthio derivatives of 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB) have been designed and synthesized from 5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole-2-thione. All compounds were evaluated for activity against human cytomegalovirus (HCMV) and/or herpes simplex virus type-1 (HSV-1). Three different cytotoxicity assays were used to determine if the compounds were toxic to uninfected cells. Most of the 2-alkylthio compounds were either inactive against HCMV and HSV-1 or were active only at concentrations at or near those which produced toxicity in uninfected cells. The best separation between activity against HCMV and cytotoxicity was observed with the 2-benzylthio analog 7. This prompted us to synthesize the substituted 2-benzylthio analogs 11-23 using a Topliss Tree approach. None of these compounds were more active than compound 7; most of the analogs were weakly active against both HCMV and HSV-1, but the activity was not separated from cytotoxicity. On the basis of both antiviral and cytotoxicity data, compound 7 was the best compound in the series. It was more active against HCMV than DRB (the 2-unsubstituted analog), acyclovir, and foscarnet, but it was less active than ganciclovir.