An in vitro colony-forming assay and flow cytometry were used to identify rat hepatoblasts as being classical MHC class I, RT1A^l-, OX18^low intercellular adhesion molecule 1 (ICAM-1)⁺. Inducible differentiation toward biliary lineage was observed in most colonies derived from single RT1A^l- progenitors, proving their bipotentiality. These findings demonstrate the antigenic profile of clonogenic hepatoblasts and proof of their bipotency. Furthermore, whereas colony formation of adult hepatocytes required epidermal growth factor, clonal growth of hepatoblasts was potentiated without epidermal growth factor. The adult hepatic colonies consisted of RT1A^l+OX18⁺ICAM-1⁺⁺ cells. These results indicate that hepatoblasts possess unique characteristics as compared with adult hepatocytes harboring significant proliferative activity. The phenotypic identity of hepatoblasts and the clonal culture system have relevance for identifying hepatic stem cells from adults, for studying liver development, and for cell therapy based on hepatic progenitors.