The incubation of a cell-free protein-synthesizing system prepared from rabbit reticulocytes with cytoplasmic RNA from herpes simplex virus (HSV)-infected cells resulted in increased thymidine kinase activity. This enzyme activity was specifically inhibited by anti-HSV antiserum and was relatively unaffected by TTP, an inhibitor of cellular thymidine kinases. Induction of the new activity was prevented by addition of inhibitors of eucaryotic protein synthesis, and no new activity was detected when RNA from cells infected with pyrimidine deoxyribonucleoside kinase-deficient mutants, instead of wild-type HSV, was added. An increased deoxycytidine kinase activity with similar properties to the HSV-specified enzyme activity was also present in cell-free systems incubated with RNA from HSV-infected cells. Phosphorylation of thymidine and deoxycytidine at 30 degrees C continued for longer than 11 h. The findings are consistent with the accurate synthesis in vitro of enzymically active HSV-specified pyrimidine deoxyribonucleoside kinase.