Two proteins, organic solute transporter (OST) and , have recently been identified that mediate bile acid export from the ileal enterocyte. It is unclear whether these two proteins associate directly and how they interact to mediate transport function and membrane localization. In this study, the protein-protein interaction, transport function and membrane localization of human OST- and proteins were examined. The results demonstrated that the co-expression of hOST- and hOST in transfected cells resulted in a 3-5 fold increase of the initial rate of taurocholate influx or efflux compared with cells expressing each protein individually and non-transfected cells. Confocal microscopy demonstrated plasma membrane co-localization of hOST- and hOST proteins in cells co-transfected with hOST- and hOST cDNAs. Protein-protein interaction between hOST- and hOST was demonstrated by mammalian two-hybrid and co-immunoprecipitation analyses. Truncation of the amino-terminal 50 amino acid extracellular residues of hOST- abolished its interaction with hOST and led to an intracellular accumulation of the two proteins and to only background levels of taurocholate transport. In contrast, carboxyl-terminal 28 amino acid truncated hOST- still interacted with hOST and majority of this cytoplasmic tail truncated protein was expressed on the basolateral membrane when stably co-transfected with hOST protein in MDCK cells. In summary, human OST- and proteins are physically associated. The intracellular carboxyl-terminal domain of human OST- is not essential for this interaction with hOST. The extracellular amino-terminal fragment of human OST- may contain important information for assembly of the heterodimer, and trafficking to the plasma membrane.