Cytokeratins are the largest subfamily of intermediate filament proteins and include more than 20 different gene products, which are expressed in an epithelial tissue-specific manner. We studied by immunohistochemistry and confocal microscopy the distribution of cytokeratin subtypes in the biliary system of adult rat liver. A cytokeratin (CK)19+/7- cholangiocyte population was observed in the smaller branches of the biliary tree including the canals of Hering. They proliferated after 2-acetaminofluorene (AAF) administration, although later the typical oval cells expressed CK7. This observation suggests that cholangiocytes with this cytokeratin phenotype may harbor adult hepatic stem cells. The CK19+/7- cholangiocytes were not present in the rat liver at birth, but developed postnatally. Similar cell populations were not observed in human livers. In conclusion, we propose that the CK19+/7- phenotype may be characteristic for adult hepatic stem cells in rat liver and that these cells are generated de novo after birth.