ABCC3 (MRP3) is a member of the family of multidrug resistance-associated proteins (MRP), which belong to the largest family of membrane transport proteins, namely, the ATP binding cassette (ABC) transporters. Members of this family contribute to the excretion of several organic anions from cells and play a critical role in conferring resistance against drugs used in the treatment of cancer. The overexpression of ABCC3 in the yeast Pichia pastoris and its subsequent purification made possible the study of substrate-dependent ATPase activity [Chloupkova, M., et al. (2007) Biochemistry 46, 7992-8003]. Here we describe the successful reconstitution of purified ABCC3 in proteoliposomes and ABCC3-dependent uptake of the anticancer drug methotrexate (MTX), as well as the physiological substrate leukotriene C(4) (LTC(4)). Our results show specific transport in a cell-free environment and in the absence of other proteins, revealing positive allosteric cooperativity for ABCC3-mediated substrate translocation. The ABCC3-mediated transport of MTX indicates a Hill coefficient of 2.3 +/- 1.7, a maximum transport rate (V(max)) of >2 micromol min(-1) mg(-1), and a K(M) in the millimolar range, whereas the translocation of LTC(4) into proteoliposomes displayed a Hill coefficient of 2.3 +/- 0.5 with a maximum transport rate of 4.7 +/- 0.8 nmol min(-1) mg(-1), and a K(M) in the micromolar range (1.7 +/- 0.3 microM). The transport of both substrates, MTX and LTC(4), was inhibited by etoposide, confirming a higher affinity of ABCC3 for LTC(4) than for MTX. The technical advances described in this report represent the basis for the extended and detailed kinetic characterization of ABCC3 with a wide range of implications for the investigation of other human ABC transporters.