Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17-estradiol 17-(-D-glucuronide) (E 2 17G), very poorly. By mutating variant residues in mrp1 to those present in MRP1, we identified Glu 1089 of MRP1 as being critical for anthracycline resistance. However, Glu 1089 mutations had no effect on E 2 17G transport. We have now identified a nonconserved amino acid within the highly conserved COOH-proximal transmembrane helix of MRP1/mrp1 that is important for transport of the conjugated estrogen. Converting Ala 1239 in mrp1 to Thr, as in the corresponding position (1242) in MRP1, increased E 2 17G transport 3-fold. Any mutation of mrp1 Ala 1239 , including substitution with Thr, decreased resistance to vincristine and VP-16 without altering anthracycline resistance. However, introduction of a second murine to human mutation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16. To confirm the importance of MRP1 Thr 1242 for E 2 17G transport and drug resistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys. These mutations decreased E 2 17G transport 2-fold. Conversion to Asp eliminated transport of the estrogen conjugate and also decreased leukotriene C 4 transport ϳ2-fold. The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested. As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/ T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C 4 and E 2 17G. These results demonstrate the important role of Thr 1242 for E 2 17G transport. They also reveal a highly specific functional relationship between nonconserved amino acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both proteins to confer similar levels of resistance to vincristine and VP-16.Human multidrug resistance protein 1 (MRP1), 1 a 190-kDa glycoprotein, is a member of the ATP-binding cassette (ABC) transporter superfamily (1-3). The predicted structure of MRP1 differs from that of a typical eukaryotic ABC transporter such as P-glycoprotein (P-gp). It contains a P-gp-like core region, which consists of two membrane-spanning domains (MSDs), each containing six transmembrane (TM) ␣-helices, and two nucleotide binding domains. However, MRP1 contains a third MSD predicted to consist of five TM ␣-helices with an extracellular NH 2 terminus and a cytoplasmic linker connecting the additional MSD with the core region (3-7). Like P-gp, MRP1 confers resistance to many commonly used, structurally diverse natural product chemotherapeutic agents including anthracyclines, Vinca alkaloids, and epipodophyllotoxins (7-10). However, several lines of evidence suggest that MRP1 and P-gp confer resistance to these drugs by different mechanisms. In vitro studies using P-gp-enriched membrane vesicles or purifi...