1-(2′-Deoxy-2′-fluoro-β- l-arabinofuranosyl)-5-methyluracil ( l-FMAU) was shown to have potent antiviral activity against Epstein-Barr virus (EBV) without any cellular toxicity at concentrations up to 200 μM (Yao et al., Biochem Pharmacol 51: 941–947, 1996). The 5′-triphosphate of l-FMAU was not a substrate for EBV or cellular DNA polymerases, but could inhibit the elongation reaction, 3′-to-5′ exonuclease activity, and nucleotide turnover catalyzed by EBV DNA polymerase. DNA synthesis catalyzed by human DNA polymerases was inhibited to a lesser extent. The inhibition pattern of EBV DNA polymerase by l-FMAU-5′-triphosphate ( l-FMAU-TP) was consistent with an uncompetitive mechanism when dNTP or template-primer were used as the variable substrates. The K i values were 38 ± 10 μM for the elongation reaction, and about 50 ± 10 μM for both nucleotide exchange and 3′-to-5′ exonuclease reactions, values that were 10–20 times less than that for GMP. l-FMAU-TP is the first nucleoside 5′-triphosphate shown to have such unique behavior toward DNA polymerases. EBV DNA polymerase could be one of the targets for the inhibitory effect of l-FMAU-TP on EBV replication.