The adult liver progenitor cells appear in response to several types of pathological liver injury, especially when hepatocyte replication is blocked. These cells are histologically identified as cells that express cholangiocyte markers and proliferate in the portal area of the hepatic lobule. Although these cells play an important role in liver regeneration, the precise characterization that determines these cells as self‐renewing bipotent primitive hepatic cells remains to be shown. Here we attempted to isolate cells that express a cholangiocyte marker from the adult mouse liver and perform single cell‐based analysis to examine precisely bilineage differentiation potential and self‐renewing capability of these cells. Based on the results of microarray analysis and immunohistochemistry, we used an antibody against CD133 and isolate CD133⁺ cells via flow cytometry. We then cultured and propagated isolated cells in a single cell culture condition and examined their potential for proliferation and differentiation in vitro and in vivo. Isolated cells that could form large colonies (LCs) in culture gave rise to both hepatocytes and cholangiocytes as descendants, while maintaining undifferentiated cells by self‐renewing cell divisions. The clonogenic progeny of an LC‐forming cell is capable of reconstituting hepatic tissues in vivo by differentiating into fully functional hepatocytes. Moreover, the deletion of p53 in isolated LC‐forming cells resulted in the formation of tumors with some characteristics of hepatocellular carcinoma and cholangiocarcinoma upon subcutaneous injection into immunodeficient mutant mice. These data provide evidence for the stem cell‐like capacity of isolated and clonally cultured CD133⁺ LC‐forming cells. Conclusion: Our method for prospectively isolating hepatic progenitor cells from the adult mouse liver will facilitate study of their roles in liver regeneration and carcinogenesis. (HEPATOLOGY 2008;48:1964‐1978.)