Transport of organic anions within hepatocytes and the possible involvement of intracellular vesicles were studied with fluorescence microscopy. For this purpose monochlorobimane, a nonfluorescent hydrophobic compound that readily permeates into cells and is conjugated with glutathione to form the fluorescent glutathione bimane, was used. In the isolated perfused livers of normal rats, glutathione bimane is rapidly secreted into bile. In contrast, in our study of livers from mutant TR- rats, a 100-fold reduction in glutathione bimane secretion into bile occurred. Mutant TR- rats have an inherited defect in the canalicular multispecific organic anion transporter, which mediates the ATP-dependent secretion of a wide range of organic anions over the canalicular membrane into bile. When cultured Wistar and TR- hepatocytes were loaded with glutathione bimane, both cell types displayed a strong cytosolic fluorescence. Wistar cells completely lost this cytosolic fluorescence at incubation on monochlorobimane-free medium because of secretion of glutathione bimane. A clear punctate fluorescence remained, however, which was scattered through the cell with some perinuclear concentration. In some cells vesicular fluorescence was also concentrated around a canaliculus. In contrast, TR- cells lost their cytosolic fluorescence more slowly and completely lacked the vesicular fluorescence. Making cells selectively permeable with digitonin directly after loading them with glutathione bimane to remove cytosolic fluorescence again revealed the presence of fluorescent vesicles in Wistar cells and their absence in TR- cells. In Wistar cells vesicular fluorescence could be increased by preincubation with monensin or methylamine, compounds that have been shown to interfere with plasma membrane recycling. In conclusion, these results suggest that apart from secretion over the plasma membrane, the canalicular multispecific organic anion transporter may be involved in accumulation of organic anion in intracellular vesicles. It is hypothesized that this intracellular localization of the canalicular multispecific organic anion transporter is caused by recycling of the transporter between the plasma membrane and intracellular membranes.