Hybridomas were produced with immune spleen cells generated by immunization of Balb/c mice with oval cell antigen (OC.2)-positive 17-day fetal liver cells isolated on antibody-coated magnetic beads. A primary screen by indirect immunofluorescence on frozen sections of fetal and adult liver identified several hybridomas secreting antibodies reactive with bile ducts and oval cells. One of these recognized an epitope, designated BD1, which was expressed on intrahepatic bile ducts in 16-day fetal and adult rat liver and in liver from rats fed a choline-deficient diet containing ethionine and rats treated with 2-acetylaminofluorine, but was absent from morphologically defined oval cells induced by a choline-deficient diet containing ethionine or by 2-acetylaminofluorine. Double-labeling immunofluorescence analysis with monoclonal antibody BD1 and OV6, a monoclonal antibody that reacts uniformly with all bile ducts and oval cells, revealed that BD1 expression on the intrahepatic bile ductular cells of normal adult rat liver was heterogeneous with a major ductal cell population (60% to 70%) expressing high levels of BD1 and a minor ductal cell population (30% to 40%) displaying undetectable or low levels of BD1 expression. Analysis of fetal liver demonstrated the presence of BD1-positive cells at day 16 of gestation on duct-like structures in contact with portal mesenchyme, an observation suggesting that expression of BD1 was associated with commitment of hepatoblasts to a ductular lineage. Taken together, our findings suggest that oval cells may be derived from an antigenically distinct subpopulation of bile ductal cells.