Multidrug resistance-associated protein 2 (Mrp2, Abcc2) is an ATP-binding cassette transporter localized at the canalicular membrane of hepatocytes that plays an important role in bile formation and detoxification. Prior in vitro studies suggest that Mrp2 can bind to Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), a PDZ protein that cross-links membrane proteins to actin filaments. However the role of NHERF-1 in the expression and functional regulation of Mrp2 remains largely unknown. Here we examine the interaction of Mrp2 and NHERF-1 and its physiological significance in HEK293 cells and NHERF-1 knock-out mice. Mrp2 co-precipitated with NHERF-1 in co-transfected HEK293 cells, an interaction that required the PDZ-binding motif of Mrp2. In NHERF-1(-/-) mouse liver, Mrp2 mRNA was unchanged but Mrp2 protein was reduced in whole cell lysates and membrane-enriched fractions to approximately 50% (p < 1 x 10(-6)) and approximately 70% (p < 0.05), respectively, compared with wild-type mice, suggesting that the down-regulation of Mrp2 expression was caused by post-transcriptional events. Mrp2 remained localized at the apical/canalicular membrane of NHERF-1(-/-) mouse hepatocytes, although its immunofluorescent labeling was noticeably weaker. Bile flow in NHERF-1(-/-) mice was reduced to approximately 70% (p < 0.001) in association with a 50% reduction in glutathione excretion (p < 0.05) and a 60% reduction in glutathione-methylfluorescein (GS-MF) excretion in isolated mouse hepatocyte (p < 0.01). Bile acid and bilirubin excretion remained unchanged compared with wild-type mice. These findings strongly suggest that NHERF-1 binds to Mrp2, and plays a critical role in the canalicular expression of Mrp2 and its function as a determinant of glutathione-dependent, bile acid-independent bile flow.