The inhibition of herpes simplex virus type 1 (HSV-1) plaque formation by acycloguanosine (ACG) was assayed in human fetal lung fibroblasts (HL), cell lines from human cervical carcinoma, rabbit cornea, and human rhabdomyosarcoma, and three green monkey kidney cell lines. The ACG concentration giving 50% plaque reduction (PR50) of HSV-1 was lowest in HL. In two green monkey kidney cell lines, HSV-1 plaque formation was relatively insensitive to ACG, with PR50 of 25 and 60 muM, respectively. In the other cells, HSV-1 showed an intermediate sensitivity to ACG. Also, HSV-2 was more sensitive to ACG in HL than in monkey kidney cells. HSV-1 plaque reduction on HL cells was studied as a function of increasing virus MOI with a constant concentration of ACG. An increased MOI decreased the sensitivity to ACG. The sensitivity of cytomegalovirus (CMV), strain Ad. 169, and four fresh CMV isolates to ACG was studied in HL cells. At low MOIs, three of the five CMV strains were somewhat sensitive to ACG, PR50 values ranging from 60 to 450 muM ACG. At high MOIs none of the virus strains were sensitive. ACG at concentrations of 200 muM or less did not significantly affect host cell DNA synthesis, as measured by 3H-thymidine incorporation. In cell culture, the inhibition of herpesviruses by ACG appears to be complex, depending on the type of virus, virus concentration, type of host cells, and condition of the cells.