Primary chimpanzee hepatocytes were maintained in vitro utilizing a serum-free medium. Hepatocyte functions were sustained throughout the culture period as demonstrated by the synthesis and secretion of liver-specific plasma proteins characteristic for differentiated hepatocytes. Hepatocyte cultures established from a chimpanzee chronically infected with human hepatitis B virus exhibited the synthesis and secretion of hepatitis B virus proteins into the medium. In addition, the de novo replication of hepatitis B virus was documented by the recovery of virus, exhibiting an endogenous DNA polymerase activity, from the tissue culture medium. Therefore, both the long-term maintenance of differentiated hepatocytes and the expression of hepatitis B virus from these primary cultures were sustained in the serum-free medium.