TR- mutant Wistar rats secrete markedly fewer organic anions other than bile acids from the liver into the bile than do control rats. Fluorescence-image analysis of isolated normal and TR- hepatocyte "doublets", which retain a bile canaliculus between them, revealed that normal hepatocytes readily transport a fluorescent bile acid (fluorescein isothiocyanate glycocholate) and a nonbile acid organic anion (carboxydichlorofluorescein diacetate) into the canaliculus. Hepatocyte doublets from TR- rats also transported fluorescein isothiocyanate glycocholate normally, but transport of carboxydichlorofluorescein diacetate into the canaliculus was negligible. Vesicles derived from the canicular domain of the plasma membrane of hepatocytes (CMV) from control and TR- rats were used to characterize the transport process for 35S-labeled bromosulphthalein and 35S-labeled bromosulphthalein glutathione, which represent nonbile acid organic anions. CMV from normal rat hepatocytes had an ATP- and temperature-dependent, saturable transport process for these 35S-labeled compounds that was absent in CMV from TR- rats. CMV from TR- rats retained normal ATP-dependent transport of daunomycin, and immunologic blots with a monoclonal antibody against the multidrug resistance gene product, P-glycoprotein, revealed no difference between normal and TR-CMV. These studies reveal that the bile canaliculus in normal rats contains an ATP-dependent organic anion transport system that is functionally absent in TR- mutant rats. The defect in TR- mutant rats is phenotypically similar to that seen in mutant Corriedale sheep and in the Dubin-Johnson syndrome in man.