Human BSEP (ABCB11) mutations are the molecular basis for three clinical forms of liver diseases, (1) progressive familial intrahepatic cholestasis type 2 (PFIC2), (2) benign recurrent intrahepatic cholestasis type 2 (BRIC2) and (3) intrahepatic cholestasis of pregnancy (ICP). To better understand the pathobiology of these disease phenotypes, we hypothesized that different mutations may cause significant differences in protein defects. Therefore we compared the effect of two PFIC2 mutations (D482G, E297G) with two BRIC2 mutations (A570T and R1050C) and one ICP mutation (N591S) with regards to the subcellular localization, maturation, and function of the rat Bsep protein. Bile salt transport was retained in all but the E297G mutant. Mutant proteins were expressed at reduced levels on the plasma membrane of transfected HEK293 cells compared to wild type (WT) Bsep in the following order: WT> N591S> R1050C~A570T~E297G>> D482G. Total cell protein and surface protein expression were reduced to the same extent, suggesting that trafficking of these mutants to the plasma membrane is not impaired. All Bsep mutants accumulate in perinuclear aggresomes-like structure in the presence of the proteasome inhibitor, MG-132 suggesting that mutations are associated with protein instability and ubiquitin-dependent degradation. Reduced temperature, sodium butyrate and sodium 4-phenylbutyrate enhanced the expression of the mature and cell surface D482G protein in HEK293 cells. These results suggest that the clinical phenotypes of PFIC2, BRIC2 and ICP may directly correlate with the amount of mature protein that is expressed at the cell surface and that strategies to stabilize cell surface mutant protein may be therapeutic.