Previous studies have identified the ATP-dependent export of glutathione conjugates as a physiological function of the multidrug resistance protein (MRP). The involvement of MRP in the transport of endogenous and xenobiotic conjugates was investigated further using membrane vesicles from MRP-transfected HeLa cells. The ATP-dependent transport of the glutathione conjugates [³H]leukotriene C₄, S-(2,4-dinitrophenyl)-[³H]glutathione, and ³H-labeled oxidized glutathione was characterized by determination of the transport efficiency V_max:K_m amounting to 1031, 114, and 7.1 ml × mg protein^-1 × min^-1, respectively. Additional endogenous substrates for MRP-mediated transport included the steroid conjugate 17β-glucuronosyl [³H]estradiol and the bile salt conjugates [6α-¹⁴C]glucuronosylhyodeoxycholate and 3α-sulfatolithocholyl [³H]taurine. The K_m value of MRP for 17β-glucuronosyl [³H]estradiol was 1.5 ± 0.3 µm, with a V_max:K_m ratio of 42 ml × mg protein^-1 × min^-1, and a K_i value of 0.7 µm for the leukotriene receptor antagonist MK 571. MRP-mediated ATP-dependent transport was observed for the anticancer drug conjugates glucuronosyl [³H]etoposide and monochloro-mono[³H]glutathionyl melphalan, but not for unmodified [¹⁴C]doxorubicin, [³H]daunorubicin, or [³H]vinblastine. Our results establish that MRP functions as an ATP-dependent export pump not only for glutathione conjugates but also for glucuronidated and sulfated endogenous as well as exogenous compounds.