Hepatitis B virus (HBV) infection is highly specific to primates. To cross the species barrier, we injected naked plasmid DNA containing a 1.5-fold-overlength genome of HBV into immunocompetent mice via the tail vein with acute circulatory overload. The injection resulted in production of viral transcripts specifically in the liver and expression of hepatitis B core protein in approximately 3% of the hepatocytes. HBV was secreted into the blood, evidenced by the presence of DNase I-resistant HBV sequence in fractionated serum at a density of 1.21g/ml. The HBV DNA positivity in the serum persisted for 1 week. Most mice became positive for hepatitis B surface antigen for 2 weeks and later seropositive for anti-hepatitis B surface antibody. This simple and efficient HBV replication system in mice could be useful for investigating whether viral mutations as well as host genetic background may affect viral replication and induction of immune response.