For the systematic analysis of various clinical and molecular aspects of hepatitis B virus (HBV) infection, an experimental small animal system of HBV infection would be a great advance. The susceptibility to HBV infection, therefore, of hepatocytes from the tree shrew species tupaia belangeri was studied in vitro and in vivo. Primary hepatocytes isolated from livers of tupaias can be reproducibly infected with HBV. In vitro infection results in viral DNA and RNA synthesis in hepatocytes and secretion hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) into culture medium. Tupaias can also be infected with HBV in vivo, resulting in viral DNA replication and gene expression in tupaia livers. Similar to acute, self-limited hepatitis B in humans HBsAg is rapidly cleared from serum, followed by seroconversion to anti-HBe and anti-HBs. These data clearly tht HBV is infectious to tupaia hepatocytes in vitro and transiently in vivo. Tupaias, therefore, may become a useful model for the experimental analysis of various molecular and clinical aspects of HBV infection, including the significance of HBV quasispecies, the steps involved in hepatocarcinogenesis as well as the evaluation of various antiviral strategies.