Acid phosphatase, alkaline phosphatase and 5'-nucleotidase activities were analyzed cytophotometrically in cryostat sections of rat liver up to 8 weeks after ligation and transsection of the common bile duct. Ligation resulted in cholestasis and induced alterations in both localization and activity of the enzyme investigated. The cellular distribution but not the activity of acid phosphatase changed in liver parenchyma. In control liver, the final reaction product was localized as discrete granules in the bile canalicular region of hepatocytes. The final reaction product was precipitated more diffusely within the cytoplasm after induction of cholestasis, most probably due to increased fragility of lysosomal membranes. In control liver, alkaline phosphatase activity was low and localized in the bile canalicular plasma membranes only. The total parenchymal activity increased threefold after the induction of cholestasis and is considered to be a compensatory mechanism in order to enhance the excretion of bile salts from hepatocytes. 5'-Nucleotidase was present at the bile canalicular and sinusoidal surfaces of plasma membranes of hepatocytes in control liver; total activity in pericentral areas was significantly higher than in periportal areas. Induction of cholestasis resulted in higher total activity and redistribution of the activity over all three surfaces of the plasma membranes, whereas heterogeneity over the different zones of the acinus disappeared. The appearance of the enzyme at lateral plasma membranes is suggested to be related to the formation of new sites for bile salt transport out of the hepatocytes. With respect to all three enzymes studied, alterations of liver parenchymal cells due to a disturbed bile transport were already established during the first week of cholestasis.