Multidrug Resistance Protein 3 (MRP3) is an ATP-dependent transporter of 17ß-estradiol 17ß(D-glucuronide)(E₂17ßG), leukotriene C₄ (LTC₄), methotrexate, and bile salts taurocholate and glycocholate. In the present study, the role of a highly conserved Trp residue at position 1242 on MRP3 transport function was examined by expressing wild-type MRP3 and Ala, Cys, Phe, Tyr and Pro substituted mutants in HEK293T cells. Four MRP3-Trp¹²⁴² mutants showed significantly increased E₂17ßG uptake whereas transport by the Pro mutant was undetectable. Similarly, the Pro mutant did not transport LTC₄. By comparison, LTC₄ transport by the Ala, Cys, Phe and Tyr mutants was reduced by ~35%. The Ala, Cys, Phe, and Tyr mutants all showed greatly reduced methotrexate and leucovorin transport, except the Tyr mutant which transported leucovorin at levels comparable to wild-type MRP3. In contrast, the MRP3-Trp¹²⁴² substitutions did not affect taurocholate transport, or taurocholate and glycocholate inhibition of E₂17ßG uptake. Thus, Trp¹²⁴² substitutions markedly alter the substrate specificity of MRP3, but leave bile salt binding and transport intact.