Deoxythymidine kinases (EC 2.7.1.—) induced in HeLa TK − cells by Herpes simplex Type I and Type II viruses both had a requirement for divalent cations. The enzymes had the highest activities in the presence of Mg 2+, followed by Mn 2+, Ca 2+, Fe 2+, and in that order, whereas they were inactive in the presence of Zn 2+ and Cu 2+. The amount of Mg 2+ required for optimal activity was dependent on the amount of ATP present, so that optimal activities were found when the concentration of Mg 2+ was equal to that of ATP; an excess of Mg 2+ inhibited the reaction. The activities of various nucleoside triphosphates as phosphate donors for Herpes simplex virus Type I deoxythymidine kinase were in the order: ATP = dATP = ara ATP > CTP > dCTP > UTP > dUTP > GTP > dGTP. Those for Herpes simplex virus Type II deoxythymidine kinase were in the order: CTP > dCTP = ara CTP > dATP > ATP > UTP > GTP > dUTP = dGTP. For both deoxythymidine kinases induced by Herpes simplex virus, the nucleoside triphosphates tested exerted cooperative effects. The K m values of ATP and CTP for the Herpes simplex virus Type I enzyme were 30 and 70 μM respectively; whereas those for the Herpes simplex virus Type II enzyme were 140 and 450 μM. Studies on binding of various thymidine analogs with free 5′-OH to these deoxythymidine kinases indicated that 5-substituted ethyl-, vinyl-, allyl-, propyl-, iodo- and bromo-dUrd as well ad iodo 5 dCyd and bromo 5 dCyd had good affinity to both enzymes. In contrast, vinyl 5 Urd, iodo 5 Urd and arabinosylthymidine had good affinity only to the Herpes simplex virus Type I enzyme but not to the Herpes simplex virus Type II deoxythymidine kinase. All of these thymidine analogs were competitive inhibitors, with K I values in the range of 0.25 to 1.5 μM. Herpes simplex virus Type I deoxythymidine kinase was less sensitive to either dTTP or iodo dUTP inhibition than Herpes simplex virus Type II. Both dThd and dCyd could serve as substrates and competed with each other for Herpes simplex viruses Type I and Type II induced kinases, but they differed in their K m values for these enzymes. The K m values of dThd and dCyd were 0.59 μM and 25 μM for Herpes simplex virus Type I deoxythymidine kinase; while they were 0.36 μM and 88 μM respectively for ther Herpes simplex virus Type II enzyme.