-L-5-Iododioxolane uracil was shown to have potent anti-Epstein-Barr virus (EBV) activity (50% effective concentration = 0.03 µM) with low cytotoxicity (50% cytotoxic concentration = 1,000 µM). It exerts its antiviral activity by suppressing replicative EBV DNA and viral protein synthesis. This compound is phosphorylated in cells where the EBV is replicating but not in cells where the EBV is latent. EBV-specific thymidine kinase could phosphorylate -L-5-iododioxolane uracil to the monophosphate metabolite. The K_m of -L-5-iododioxolane uracil with EBV thymidine kinase was estimated to be 5.5 µM, which is similar to that obtained with thymidine but about fivefold higher than that obtained with 2' fluoro-5-methyl--L-arabinofuranosyl uracil, the first L-nucleoside analogue discovered to have anti-EBV activity. The relative V_max is seven times higher than that of thymidine. The anti-EBV activity of -L-5-iododioxolane uracil and its intracellular phosphorylation could be inhibited by 5'-ethynylthymidine, a potent EBV thymidine kinase inhibitor. The present study suggests that -L-5-iododioxolane uracil exerts its action after phosphorylation; therefore, EBV thymidine kinase is critical for the antiviral action of this drug.