Absence of a functional multidrug resistance protein 2 (MRP2; symbol ABCC2) from the hepatocyte canalicular membrane is the molecular basis of Dubin- Johnson syndrome, an inherited disorder associated with conjugated hyperbilirubinemia in humans. In this work, we analyzed a relatively frequent Dubin-Johnson syndrome mutation that leads to an exchange of two hydrophobic amino acids, isoleucine 1173 to phenylalanine (MRP2I1173F), in a predicted extracellular loop of MRP2. HEK-293 cells stably transfected with MRP2I1173F cDNA synthesized a mutant protein that was mainly core-glycosylated, predominantly retained in the endoplasmic reticulum, and degraded by proteasomes. MRP2I1173F did not mediate ATP-dependent transport of leukotriene C₄ (LTC₄) into vesicles from plasma membrane and endoplasmic reticulum preparations while normal MRP2 was functionally active. Human HepG2 cells were used to study localization of MRP2I1173F in a polarized cell system. Quantitative analysis showed that GFP-tagged MRP2I1173F was localized to the apical membrane in only 5% of transfected, polarized HepG2 cells compared with 80% for normal MRP2-GFP. Impaired protein maturation followed by proteasomal degradation of inactive MRP2I1173F explain the deficient hepatobiliary elimination observed in this group of Dubin-Johnson syndrome patients.