SUMMARYBHK (dPyK-) cells infected with herpes simplex virus type 1 (HSV-1) contain a virus-induced deoxythymidine (dThd)-phosphorylating enzyme. This enzyme uses AMP as phosphate donor and is called AMP:deoxythymidine 5'-phosphotransferase (or kinase). The enzyme was purified over 1300-fold and was found to be specific for an AMP substrate. It can thus be distinguished from virus-specific deoxypyrimidine kinase (dPyK). It was shown that the two substrates AMP and dThd participate in the reaction at a 1 : 1 molar ratio; the K m for AMP was 2.3 gM and for dThd it was 2, 1 #M. The mol. wt. of the enzyme was estimated to be between 110000 (by glycerol gradient centrifugation) and 90000 (by gel filtration). For optimum activity, the phosphotransferase required an alkaline pH, and 37 °C; the activation energy of the reaction was 18 450 cal/mol. The appearance of the enzyme after infection parallels that of viral DNA synthesis-related functions.