L-nucleoside analogs are a new class of antiviral and anticancer agents, several of which are currently used in the clinic. The phosphorylation of these agents to the triphosphate form is thought to be important for exertion of their pharmacological activities. 1-(2'-deoxy-2'-fluoro-beta-L-arabinofuranosyl)-5-methyluracil (L-FMAU; Clevudine) is a thymidine analog that is currently under phase III clinical trials as an anti-human hepatitis B virus agent. We examined the behavior of its monophosphate metabolite with human recombinant thymidylate kinase (TMPK) and showed that L-FMAU monophosphate (L-FMAUMP) is a poorer substrate than its D-configuration anomer (D-FMAUMP). The phosphorylation efficiency of l-FMAUMP is similar to that of the monophosphate of 2',3'-didehydro-2',3'-dideoxythymidine (d4T), an anti-human immunodeficiency virus analog, both of which are approximately 1% TMP. To clarify the role of human TMPK in the phosphorylation of L-FMAUMP to the diphosphate metabolite in cells, a Tet-On inducible human TMPK cell line system was established. In this system, the expression of TMPK is closely regulated in response to various concentrations of doxycycline. When the cells were treated with L-FMAU or d4T, the amounts of the diphosphate and triphosphate metabolites of these analogs were increased, in accordance with an increase in human TMPK activity in cells. In conclusion, this is the first demonstration of the behavior of TMPK toward L-FMAUMP. This study indicates that human TMPK can phosphorylate L-FMAUMP and play a critical role in L-FMAU metabolism in cells.