Hybrids were created by fusion of primary rat hepatocytes with well-differentiated Reuber H35 rat hepatoma cells. Seventeen hybrids were screened for bile acid synthesis using [26-14C]cholesterol. As [26-14C]cholesterol was converted to bile acid, 14CO2 was released. Using this assay, four hybrids (8B, 12C, 13C, and 13D) were identified which synthesized bile acid. These four hybrids also incorporated [14C]taurine into bile acid. Bile acids were identified by capillary gas chromatography/mass spectrometry, and their rates of synthesis were quantitated by isotope dilution. Reuber H35 cells synthesized little or no bile acid. However, hybrids 8B, 12C, 13C, and 13D synthesized chenodeoxycholic acid, alpha-muricholic acid, and cholic acid and secreted them into the media. The rates of synthesis of individual bile acids varied among these hybrids. For example, the relative percentage of cholic acid ranged from 11.1% (hybrid 8B) to 50.4% (hybrid 13C). The bile acids synthesized and secreted by the most active hybrid, 12C, were greater than 93% conjugated. In summary, hybrids were created that retain the capacity to synthesize, conjugate, and secrete three major rat bile acid species. Such hybrids are unique model systems that will allow the study of the biochemical and genetic regulation of bile acid synthesis.