Thymidine kinase UL23 gene (EC 2.7.1.145) from the L2 acyclovir-sensitive strain of herpes simplex virus type 1 was cloned and expressed in E. coli. The enzyme was purified by chromatography to the purity of 90% according to PAG electrophoresis data. The Michaelis constants for the reactions with thymidine and acyclovir were determined. The enzyme was found to phosphorylate modified nucleosides, particularly 3'-deoxythymidine, 3'-deoxy-2',3'-didehydrothymidine, 2',3'-dideoxycytidine, 9-[(hydroxyethyl)methyl]guanine, E-5-(2-bromovinyl-2'- deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, 2',3'- dideoxydehydrothymidine, β-L-2',3'-dideoxy-3'-thiacytidine, and 3'-fluoro-3'-deoxythymidine. Some properties of the purified enzyme were compared with those of thymidine kinases of other herpes simplex virus strains. It was shown that acyclovir H-phosphonate inhibited the enzyme. © Pleiades Publishing, Ltd., 2011.