The ductular reaction to acute submassive necrosis was studied in human livers removed at the time of orthotopic liver transplantation. Single, double, and triple immunohistochemical labeling in combination with morphometry was used to analyze the phenotype and proliferative and apoptotic rates of various epithelial cell compartments. These were divided on the basis of immunohistochemistry and morphology into three subtypes: 1) CK19+/AE1+ mature bile duct epithelium, 2) HEP-PAR+ mature hepatocytes (HEPs), and 3) CK19+/AE1+ ductular hepatocyte (DH) cells lying at the interface between the portal tract connective tissue and the hepatic lobules. Cycling cells were defined as those showing Ki-67+(MIB-1) nuclear labeling. Apoptotic cells were identified with in situ labeling using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling assay. Special emphasis was placed on DHs that appeared at the interface between the portal tracts and hepatic lobules. During the recovery phase from submassive hepatic necrosis, subtraction of the rate of cell death from the proliferative index shows that all of the epithelial compartments experience a net increase in the number of cells. The highest proliferation rate occurs in the DHs, which is significantly (P< 0.0001) higher than the proliferation rate seen in either the HEP or bile duct epithelium compartments. Immunohistochemical analysis of the highly proliferative DH compartment shows it to be a heterogeneous population with unique phenotypic features. Like epithelial cells in the ductal plate of fetal liver and cholangiocarcinomas, DHs are positioned on a laminin-rich matrix and focally express vimentin and Lewis (x) and show up-regulation of bcl-2 and type IV collagenase. However, unlike ductal plate cells, DHs are CD34 and alpha-fetoprotein negative. Although a subpopulation of DHs share phenotypic features with mature bile duct epithelium (AE1/cytokeratin 19 and type IV collagenase positive) or HEP (HEP-PAR, albumin, and alpha-1-antitrypsin positive), they are also clearly separate from both populations; DHs are negative or only weakly stain for glutathione-S-transferase-pi and are type IV collagenase positive. Moreover, occasional DHs also co-expressed HEP-PAR or alpha-1-antitrypsin and AE1, indicative of both hepatocyte and ductular differentiation. These findings suggest that DHs seen in human livers after submassive necrosis may represent a transient amplifying population arising from a progenitor population located in or near the canals of Herring. In addition, injured hepatocytes can express cytokeratin 19 and AE1, which normally are biliary intermediate filaments.