Rat embryo fibroblasts (REF) morphologically transformed by herpes simplex virus type 2 (HSV-2) and tumor-derived cells were tested for ability to grow in the presence of 9-(2-hydroxyethoxymethyl) guanine (acyclovir). Results indicated that the effective dose of acyclovir (ACV) required to inhibit HSV-2-transformed and tumor-derived cell growth by 50% (ED50) compared to mock-treated control cells averaged 15 to 75 micrograms/ml. In contrast, the ED50 of acyclovir was more than HEp-2 cells. HSV-2-transformed and tumor-derived cells after both low (less than 30) and high (greater than 30) serial passages expressed detectable levels of the virus-coded thymidine kinase (TK) measured in cell extracts by serum neutralization assay. HSV-2-transformed or tumor-derived cells converted two- to ten-fold more acyclovir to phosphorylated forms than nontransformed REF cells. Preliminary data showed that the drug inhibited tumor development in newborn syngeneic rats inoculated with HSV-2-transformed cells. The inhibitory activity of acyclovir and presence of low levels of HSV-2 TK activity appeared to correlate.