Susceptibility testing of herpes simplex type 1 (HSV-1) and type 2 (HSV-2) viruses to cytosine arabinoside (Ara-C) has been standardized under conditions of optimal reproducibility, susceptibility, and simplicity. Standard conditions were defined as virus inoculum of 10(1.5) to 10(2.5) TCID(50), WI-38 strain of lung fibroblasts, 2-day incubation, and observation of virus cytopathic effect after staining by the May-Greenwald-Giemsa method. The variability of minimum inhibitory concentration (MIC) of cytopathic effect was fourfold within each test and sixfold in successive assays. MIC values of all seven tested HSV-1 strains were in the range of 0.25 to 0.75 mug per ml, whereas four of nine HSV-2 strains had MIC values of 0.9 mug per ml and greater. A similar method can be used for assay of virus inhibitory titers of body fluids.