Recent studies, including our own, suggest that intermediate filament proteins, particularly bile duct-specific cytokeratin 19 (CK19) and the hepatocyte-specific HepPar1 antigen define the developmental stages of hepatic progenitor cells during liver morphogenesis. We hypothesized that the HepPar1+ CK19+ progenitor cells are activated during human liver regeneration after massive hepatic necrosis and proliferate with the formation of so-called ductular hepatocytes or neocholangioles. We demonstrated previously that the ductular hepatocytes proliferate and share phenotypic characteristics with hepatocytes and biliary epithelial cells. In this investigation, we compared the expression pattern of intermediate filament proteins and HepPar1 antigen in ductular hepatocytes with that of bipotential hepatic progenitor cells. CK14, CK19, vimentin, and HepPar1 antigen were localized by immunoperoxidase staining in 13 human livers with regeneration after massive hepatic necrosis. Double immunostaining of three cases for CK14/CK19 and HepPar1/CK19 was also performed. CK19 reaction exhibited diffuse staining of the cytoplasm of many ductular hepatocytes and bile ducts in all cases. CK14 was expressed in the cytoplasm of ductular hepatocytes and few bile ducts in 5 of 12 specimens. HepPar1 staining was positive in many ductular hepatocytes in 11 of 13 cases. Vimentin was detected in the perinuclear cytoplasm of ductular hepatocytes and some bile duct epithelial cells in all regenerating livers. Double immunostaining for HepPar1/CK19 demonstrated that the ductular hepatocytes contained either HepPar1 or CK19 and that some ductular hepatocytes coexpressed both antigens. CK14, CK19, vimentin, and HepPar1 expression in ductular hepatocytes in human liver regeneration resembles the pattern seen in the developing human liver from 4 to 16 weeks' gestation. This suggests that the ductular hepatocytes recapitulate the developmental stages of bipotential liver progenitor cells and differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics.