Mouse and rat hepatocytes were investigated for replicative potential in primary culture under defined conditions. Mouse hepatocytes replicated at least twice or three times by the 7th day of culture in response to EGF, while only a half of rat hepatocytes replicated once. Thus a far greater potential for proliferation was demonstrated by mouse than rat cells, although the mouse cells ceased dividing by the 10th day of culture. Immunocytochemical examination revealed that the cells produced both albumin and alpha-fetoprotein. Ultrastructural investigation revealed that these cells were immature hepatocytes in nature, based on the presence of microbodies with nucleoids. Thus, primary cultures of mouse hepatocytes seem to be a useful system for studying proliferation and transformation of hepatocytes in vitro.