Various anti-viral agents, e.g., interferon, have recently been used for the treatment of viral hepatitis. In the present study, duck hepatitis B virus (DHBV) was cultured in vitro and the anti-DHBV effects of acyclovir (ACV) and vidarabin 5′-monophosphate (VMP) were studied. The portal perfusion method was applied to the livers of 7-day-old white ducks weighing 100g, bred in Japan, and hepatocytes were infected with DHBV in vitro. Duck hepatocytes infected with DHBV were cultured in medium containing ACV or VMP, and the anti-DHBV effects of these drugs were assessed by determining DHBV-DNA and duck hepatitis B surface antigen in the medium. Both ACV and VMP had anti-DHBV effects when used immediately after infection; however, both drugs were ineffective in hepatocytes obtained from a DHBV carrier duck. In conclusion, the anti-DHBV effects of these drugs were very limited. However, this culture system appears to be useful for studies of hepatitis virus and anti-viral drugs.