5-(2-Bromovinyl)-2'-deoxyuridine (BrVdUrd) is a potent and selective inhibitor of herpes simplex virus Type I (HS-I) and varicella zoster (VZ) virus replication but is much less potent against herpes simplex virus Type II (HS-II) replication. A possible enzymatic basis for this difference is reported here to involve the virus-coded dThd-dTMP kinases (EC 2.7.1.75) from the three virus strains. The thymidine kinases from the three virus strains were purified by affinity chromatography. In addition to catalyzing the phosphorylation of nucleosides, each of the three purified enzymes catalyzed the phosphorylation of thymidylate to its diphosphate but at strikingly different rates. The relative amounts of extractable virus-coded thymidylate kinases were estimated to be 100/2/40 for cells infected with HS-I, HS-II, and VZ viruses, respectively. Extracts of cells infected with HS-I virus catalyzed the phosphorylation of the monophosphate of BrVdUrd to its diphosphate. In contrast, the product was not detected with extracts from cells infected with HS-II virus. THe ratios of rates with 0.5 mM BrVdUrd monophosphate versus 0.1 mM dTMP as substrates for each of the purified dThd-dTMP kinases from HS-I, HS-II, and VZ viruses and the dTMP kinase from host cells were, respectively, 0.09, less than 0.002, 0.03, and less than 0.0002. These observations correlate with the relative sensitivities of these viruses to BrVdUrd in cell culture and suggest that, if BrVdUrd exerts its effect as a triphosphate, the inefficient phosphorylation of the monophosphate contributes to the insensitivity of HS-II virus to this agent.