Liver regeneration after partial hepatectomy results primarily from the simple division of mature hepatocytes. However, during embryonic and fetal development or in circumstances under which postnatal hepatocytes are injured, organ regeneration is believed to occur from a compartment of epithelial liver stem or progenitor cells with biliary and hepatocytic bipotentiality. The ability to identify, isolate, and transplant epithelial liver stem cells from fetal liver would greatly facilitate the treatment of hepatic diseases currently requiring orthotopic liver transplantation. Here we report the identification and immortalization by retrovirus-mediated transfer of the simian virus 40 large T antigen gene of primate fetal epithelial liver cells with a dual hepatocytic biliary phenotype. These cells grow indefinitely in vitro and express the liver epithelial cell markers cytokeratins 8/18, the hepatocyte-specific markers albumin and alpha-fetoprotein, and the biliary-specific markers cytokeratins 7 and 19. Bipotentiality of gene expression was confirmed by clonal analysis initiated from single cells. Endogenous telomerase also is expressed constitutively. After orthotopic transplantation via the portal vein, approximately 50% of the injected cells integrated into the liver parenchyma of athymic mice without tumorigenicity. Three weeks after transplantation, cells having seeded in the liver parenchyma expressed both albumin and alpha-fetoprotein but had lost expression of cytokeratin 19. These results provide strong evidence for the existence of a bipotent epithelial liver stem cell in nonhuman primates. This unlimited source of donor cells also should enable the establishment of a model of allogenic liver cell transplantation in a large animal closely related to humans and shed light on important questions related to liver organogenesis and differentiation.