Infection with HBeAg and HBV DNA positive serum in primary cultures of cryopreserved human hepatocytes in the presence of human whole blood in the medium was performed in 8 consecutive experiments. HBsAg and HBV DNA release into the medium was increased in the second week after infection. Via immunostaining, HBcAg was first observed in the nucleus of hepatocytes approximately 3 days after infection. A maximal percentage of HBcAg positive cells in 0.1% of cultured hepatocytes was detected on the 7th day. HBsAg was also first demonstrated on the 3rd day, and predominantly localized in the cytoplasm. About 5% of hepatocytes were HBsAg positive on the 12th day after infection. The percentage of positive cells did not appear to increase after this time. Using in situ cytohybridization and agarose gel electrophoresis and Southern blot analysis, HBV DNA was first detected on the 4th day. In addition, electron microscopic studies revealed the presence of 42 nm virus-like particles in the cytoplasm of infected cells in the second week after infection. This in vitro system provides a model for studying the mechanism of HBV infection, viral replication and maturation. However, further improvement of culture systems is needed, to increase the number of infected cells and for active HBV replication.