Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare liver disease characterized by early onset of cholestasis that leads to cirrhosis and liver failure before adulthood. PFIC3 may be improved by chronic administration of ursodeoxycholic acid, although in many cases liver transplantation is the only therapy. The disease is caused by mutations of the adenosine triphosphate (ATP)–binding cassette, sub-family B, member 4 (ABCB4) [multidrug resistance 3 (MDR3)] gene encoding a specific hepatocellular canalicular transporter involved in biliary phosphatidylcholine secretion. Several mutations have been reported; however, the effect of individual mutations has not been investigated. ABCB4 is highly homologous to ATP-binding cassette, sub-family B, member 1 (ABCB1) (MDR1), the multidrug transporter responsible for drug resistance of cancer cells. We have studied the effect of mutation I541F localized to the first nucleotide-binding domain, which is highly conserved between ABCB4 and ABCB1. Plasmids encoding the wild-type human ABCB4 or rat ABCB1–green fluorescing protein (GFP) construct, and corresponding I541F-mutants, were expressed in hepatocellular carcinoma, human (HepG2) and Madin-Darby canine kidney (MDCK) cells. Expression studies showed that ABCB4 was localized at the bile canalicular membrane in HepG2 cells and at the apical surface in MDCK cells, whereas the I541F mutant was intracellular. In MDCK cells, ABCB1-I541F also accumulated intracellularly in compartments, which were identified as the endoplasmic reticulum and cis-Golgi, and remained partially endoH-sensitive. After shifting cells to 27°C, ABCB1-I541F was expressed at the apical cell surface in a mature and active form. Similarly, ABCB4 was significantly trafficked to the membrane of bile canaliculi in HepG2 cells. Conclusion: Mutation I541F causes mislocalization of both ABCB4 and ABCB1. Intracellular retention of ABCB4-I541F can explain the disease in PFIC3 patients bearing this mutation. The observation that plasma membrane expression and activity can be rescued by low temperature opens perspectives to develop novel therapies for the treatment of PFIC3. (HEPATOLOGY 2009.)