To generate a model of human hepatitis B infection (HBV) in immunocompetent rats with chimeric human liver.Normal rats were tolerized to human hepatocytes by exposure to human hepatocytes at day 17 of gestation. We transplanted human hepatocytes and inoculated HBV into the rats after birth. Mixed lymphocytes assay, a measure for tolerance, indicated that animals fetally exposed to human hepatocytes developed tolerance to human hepatocytes. Spleen lymphocytes from tolerized animals did not proliferate when challenged with donor human hepatocytes. In contrast, control animals given saline fetally developed no tolerance to human hepatocytes. Tolerant animals with transplanted human hepatocytes were susceptible to HBV infection. Western blot analysis and immuno-histochemistry of liver sections from tolerized, HBV infected animals with transplanted human hepatocytes showed the presence of functioning human hepatocytes that synthesized human albumin, of which 30% were also positive for HB surface antigen and HBV DNA. The presence of covalent closed circular HBV DNA in the liver indicated active HBV viral replication.Tolerized rats with chimeric human livers can be infected with HBV and used as an animal model for HBV infection. Tolerized rats with chimeric human livers can also be used for generating models of other human hepatic viral diseases.