Inhibition by acycloguanosine (ACG) of plaque formation by harpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus, and cytomegalovirus was studied. Seventeen clinical isolates of HSV-1 were inhibited by ACG at a mean 50% inhibitory dose (ID(50)) of 0.15 +/- 0.09 muM. The mean ID(50) for 10 isolates of HSV-2 was 1.62 +/- 0.76 muM, and for four isolates of varicella-zoster virus it was 3.75 +/- 1.30 muM. The ID(50)'s for two cytomegalovirus isolates were 100 and 160 muM, and for four additional isolates of cytomegalovirus no end point (ID(50)) was reached at 200 muM. ACG at a concentration of 200 muM had no effect on deoxyribonucleic acid synthesis in human fibroblast cells and only inhibited thymidine incorporation by Vero cells by one-third. These studies demonstrated the antiviral activity of ACG against clinical isolates of HSV-1, HSV-2, and varicella-zoster virus and the lack of toxicity to monkey or human cells in culture at concentrations which markedly inhibited these viruses. ACG had little effect on cytomegalovirus at concentrations in excess of 100 muM.