A number of ABC transporter proteins are involved in the phenomenon of cancer multidrug resistance. These integral membrane proteins actively extrude a variety of cytotoxic agents, used in cancer chemotherapy. The major physiological role of the multidrug transporters is most probably the protection of our cells and tissues against toxic xenobiotics. MDR (P-glycoprotein) type ABC transporters preferentially extrude large hydrophobic, positively charged compounds, while the members of the multidrug resistance protein (MRP) family can extrude both hydrophobic and water-soluble anionic compounds. The ABCG2 transporter has a substrate specificity largely overlapping that of MDR1. By using in vitro expression systems we have studied the effects of various structural alterations on the function of human multidrug resistance proteins. In the present communication I will present data of our recent work related to the molecular dissection of the MRP1 protein. MRP1, in addition to an MDR1-like core, contains an N-terminal membrane-bound (TMD0) region and a cytoplasmic linker (L0), both characteristic of several members of the MRP family. In order to study the role of the TMD0 and L0 regions, we have constructed various truncated and mutated MRP1, and chimeric MRP1-MDR1 molecules, which were expressed in insect (Sf9) and polarized mammalian (MDCKII) cells. The function of the expressed proteins was examined in isolated membrane vesicles by measuring the transport of leukotriene C4 and other glutathione conjugates, and by vanadate-dependent nucleotide occlusion. Cellular localization, glutathione-conjugate and drug transport were also studied in MDCKII cells. We found that chimeric proteins, consisting of N-terminal fragments of MRP1 fused to the N-terminus of MDR1, preserved the transport, nucleotide occlusion, and apical membrane routing of wild-type MDR1. MRP1 without the TMD0L0 region was non-functional and localized intracellularly. Co-expression of L0 with the core region resulted in a functional MRP1 molecule, with a proper routing to the lateral membrane. Interestingly, the L0 peptide was found to be associated with membranes in Sf9 cells and could only be solubilized by urea or detergent. A ten amino acid deletion in a predicted amphipathic region of L0 abolished its attachment to the membrane, eliminated MRP1 transport function, but did not affect membrane routing. These experiments suggest that the L0 region forms a distinct domain within MRP1, which interacts with hydrophobic membrane regions and with the core region of MRP1.