The multidrug resistance-associated protein (MRP) family plays a major role in the hepatic excretion of organic anions. The expression, localization, and function of Mrp2 (Abcc2), a canalicular multispecific organic anion transport protein, were studied in sandwich-cultured rat hepatocytes. The amount of Mrp2 protein remained constant in sandwich-cultured rat hepatocytes over 4 days in culture, but the molecular mass increased approximately 10 kDa from 190 to 200 kDa. Mrp2 was internalized initially after hepatocyte isolation and was gradually sorted to the canalicular membrane. Disposition of 5-(6)-carboxy-2',7'-dichlorofluorescein (CDF), an Mrp2 substrate, confirmed the changes in Mrp2 localization. CDF was localized predominantly inside hepatocytes at day 0 and gradually localized to the canalicular domain over time in culture. By day 4 in culture, CDF was localized exclusively in the canalicular networks. Tunicamycin, an inhibitor of glycosylation, decreased the molecular mass and simultaneously impaired the trafficking of Mrp2 to the canalicular membrane. Treatment of lysates from both day 0 (Mrp2, 190 kDa) and day 4 (Mrp2, 200 kDa) sandwich-cultured rat hepatocytes with peptide N-glycosidase F, a deglycosylation agent, resulted in a band of 180 kDa, suggesting that Mrp2 from both day 0 and day 4 was glycosylated, but Mrp2 on day 4 was more glycosylated than on day 0. In conclusion, these data support the hypothesis that glycosylation of Mrp2 is responsible for the increase in molecular mass and may be involved in directing the canalicular localization of Mrp2 in sandwich-cultured rat hepatocytes over days in culture.