Hepatobiliary elimination of many organic anions is initiated by OATP1B1 (OATP2, LST-1, OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), which are the predominant uptake transporters of human hepatocytes. Thereafter, the unidirectional efflux pump ABCC2 (multidrug resistance protein 2) mediates the transport of organic anions, including glutathione conjugates and glucuronosides, into bile. In this study, we generated a Madin-Darby canine kidney (MDCKII) cell line stably expressing recombinant OATP1B1, OATP1B3, and OATP2B1 in the basolateral membrane and ABCC2 in the apical membrane. Double-transfected MDCKII cells stably expressing ABCC2 together with OATP1B1, OATP1B3, or OATP2B1 served as control cells. The quadruple-transfected cells exhibited high rates of vectorial transport of organic anions, including bromosulfophthalein, cholecystokinin peptide (CCK-8), and estrone 3-sulfate. The quadruple-transfected cells enabled the identification of substrates for uptake or vectorial transport that may be missed in studies with a double-transfected cell line, as exemplified by CCK-8, which is a substrate for OATP1B3 but not for OATP1B1 or OATP2B1. The broad substrate spectrum covered by the three hepatocellular OATP transporters enables representative analyses of the uptake of many organic anions into human hepatocytes. The broad spectrum of organic anions transported vectorially by the quadruple-transfected cells also provides valuable information on the substrate selectivity of ABCC2, without the need for studies in inside-out membrane vesicles containing the ABCC2 protein. The quadruple-transfected MDCKII-ABCC2/OATP1B1/1B3/2B1 cells may thus be useful for the identification of substrates and inhibitors, including drug candidates, undergoing uptake and secretion by human hepatocytes, under conditions that may be better defined than in primary cultures of human hepatocytes.