The architectural framework of the pericanalicular sheath composed of cytokeratin intermediate filaments (IFs) was examined after phalloidin treatment, bile duct ligation, and alcoholic fatty liver in rats to assess the role of IFs in experimental cholestasis. Electron microscopy examination of whole mount unembedded extracted liver slices was employed to visualize the cytoskeleton. Immunofluorescence staining and immunoelectron microscopy of the sheath were also performed using monoclonal antibodies to rat hepatocyte cytokeratins CK49 and CK55. The thickness of the wall and the diameter of the lumens were measured. In the phalloidin-treated rats, the pericanalicular sheath was markedly dilated and thickened. Immunofluorescence staining showed that the CK49 and CK55 IFs were localized in the pericanalicular region, particularly in the pericentral area. Immunoelectron microscopy documented that the IFs at the thickened pericanalicular sheath consisted of both CK49 and CK55, which means that the thickening of the bile canaliculus was in part due to an increase of IFs and not just due to an increase in actin filaments. In the livers where the bile duct was ligated, the pericanalicular sheath was irregularly dilated and some parts of the sheath appeared thinned out or missing. The belt desmosome also appeared absent focally in the pericanalicular sheath. Immunofluorescence studies showed that the staining for CK49 and CK55 was reduced focally in the pericanalicular region. The CK55 antibody stained the cytoplasm of hepatocytes in the periportal area more intensely when compared with the controls. These results indicated that the pericanalicular sheath and the belt desmosome were focally disrupted in response to extrahepatic bile duct obstruction. In the ethanol-fed rats, the pericanalicular sheath was dilated, thickened and tortuous, and appeared focally flattened by large fat droplets. IFs in the cytoplasm were pushed to the cell periphery and were compressed against each other by the fat droplets. CK55 and CK49 appeared increased as indicated by the observed immunofluorescence at the pericanalicular region. Immunoelectron microscopy showed that IFs of the thickened pericanalicular sheath were composed of CK55 and CK49. It is suggested that the pericanalicular sheath functions to mechanically provide a scaffolding for the bile canaliculus which is vulnerable to the different forces involved in cholestasis of different pathogenesis such as focal compression and distortion by fat, hypertrophy in response to increased F actin and focal destruction by increased intracanalicular pressure.