The antiviral activity of 2′-fluoro-5-methyl-β-l-arabinofuranosyluracil (l-FMAU), a novel l-nucleoside analog of thymidine known to be an inhibitor of hepatitis B virus (HBV) replication in hepatoma cells (2.2.1.5 cell line), was evaluated in the duck HBV (DHBV) model. Short-term oral administration (5 days) ofl-FMAU (40 mg/kg of body weight/day) to experimentally infected ducklings induced a significant decrease in the level of viremia. This antiviral effect was sustained in animals when therapy was prolonged for 8 days. The histological study showed no evidence of liver toxicity in the l-FMAU-treated group. By contrast, microvesicular steatosis was found in the livers of dideoxycytidine-treated animals. l-FMAU administration in primary duck hepatocyte cultures infected with DHBV induced a dose-dependent inhibition of both virion release in culture supernatants and intracellular viral DNA synthesis, without clearance of viral covalently closed circular DNA. By using a cell-free system for the expression of an enzymatically active DHBV reverse transcriptase, it was shown that l-FMAU triphosphate exhibits an inhibitory effect on the incorporation of dAMP in the viral DNA primer. Thus, our data demonstrate that l-FMAU inhibits DHBV replication in vitro and in vivo. Long-term administration of l-FMAU for the eradication of viral infection in animal models of HBV infection should be evaluated.