The normal surface anatomy of rat hepatocytes, sinusoids, and bile canaliculi was examined by scanning electron microscopy following finger fracture of tissues that were perfused via the portal vein and fixed with 2.5% glutaraldehyde. These normal findings were contrasted with several models of cholestasis, including infusion of sodium taurolithocholate (TLC), bile duct ligation, and ethynyl estradiol treatment, in order to examine the effects of cholestasis on morphology of the bile canaliculus. TLC-induced cholestasis was associated with specific dilation and disruption of canalicular membranes, which corresponded to evaginations of pericanalicular ectoplasm observed by transmission electron microscopy, and suggested a direct toxic effect of the bile salt on membrane structure. Infusions of lithocholate sulfate or simultaneous infusions of TLC and sodium taurocholate prevented these severe abnormalities from occurring. Bile duct ligation produced progressive canalicular dilation (from 1 to 96 hr) and loss of microvilli, but did not result in the type of membrane changes produced by TLC. No significant changes in canalicular morphology were observed following ethynyl estradiol treatment. These studies demonstrate that scanning electron microscopy is a useful technique for the evaluation of both normal and abnormal hepatic surface morphology, and indicate that TLC produces a distinctive abnormality in the structure of the canalicular membrane, which was not seen in other models of cholestasis.