Multidrug resistance-associated protein (MRP)1 and canalicular multispecific organic anion transporter (cMOAT)/MRP2 are ATP-binding cassette (ABC) transporters that confer resistance to natural product cytotoxic drugs. We recently described the complete coding sequences of four human MRP/cMOAT subfamily members and found that, among these proteins, MRP3/MOAT-D is most closely related to MRP1 (58% identity; M. G. Belinsky and G. D. Kruh, Br. J. Cancer, 80: 1342-1349, 1999). In the present study, we sought to determine whether MRP3 is capable of conferring resistance to cytotoxic drugs. To address this question, human embryonic kidney 293 cells were transfected with an MRP3 expression vector, and the drug resistance phenotype of the transfected cells was analyzed. The MRP3-transfected cells displayed approximately 4-fold resistance to etoposide and approximately 2-fold resistance to vincristine, compared with control transfected cells. In addition, approximately 1.7-fold resistance was observed for the antimetabolite methotrexate. Increased resistance was not observed for several other natural product agents, including anthracyclines and Taxol. The MRP-transfected cells exhibited reduced accumulation of radiolabeled etoposide, consistent with the operation of a plasma membrane efflux pump. These results indicate that MRP3 confers resistance to some anticancer agents but that its resistance pattern is distinct from the resistance patterns of other ABC transporters involved in resistance to natural product chemotherapeutic agents.