Multidrug resistance protein 3 (MRP3) is an ATP-dependent transporter of 17-estradiol 17(D-glucuronide) (E₂17G), leukotriene C₄ (LTC₄), methotrexate, and the 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 human embryonic kidney 293T cells. Four MRP3-Trp¹²⁴² mutants showed significantly increased E₂17G 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 with wild-type MRP3. In contrast, the MRP3-Trp¹²⁴² substitutions did not significantly affect taurocholate transport or taurocholate and glycocholate inhibition of E₂17G uptake. Thus Trp¹²⁴² substitutions markedly alter the substrate specificity of MRP3 but leave bile salt binding and transport intact.