A cell fraction enriched in biliary epithelial cells (BEC) has been isolated from the liver of normal rats. The procedure involved proteolytic digestion by trypsin and mild mechanical disruption of biliary ductular and connective tissue that remained undigested after collagenase-hyaluronidase perfusion. An adherence procedure removed the large majority of contaminating Kupffer cells. The majority (87.4 +/- 3.5%) of the cells were positive to an indirect immunofluorescence staining that used an antiserum against bovine hoof prekeratin that specifically recognizes intermediate filaments of biliary epithelium. Similar results were obtained by histochemical staining for gamma-glutamyltranspeptidase activity. The contamination of the BEC fraction with Kupffer cells and hepatocytes was approximately 7% and 2%, respectively. The viability of the BEC population was always more than 90%. The BEC and hepatocytes were analysed for their lipid composition; the BEC were found to have a cholesterol content approximately 6-times higher than hepatic parenchymal cells, with a cholesterol/phospholipid molar ratio of 0.53 in comparison to a value of 0.11 for hepatocytes. No detectable evidence of cytochrome P-450 or cytochrome P-450-related enzymatic activities was found in the BEC.