The yeast cadmium factor gene (YCF1) from Saccharomyces cerevisiae, which was isolated according to its ability to confer cadmium resistance, encodes a 1,515 amino acid ATP-binding cassette (ABC) protein with extensive sequence homology to the human multidrug resistance-associated protein (MRP1) (Szczypka, M., Wemmie, J. A., Moye-Rowley, W. S., and Thiele, D. J. (1994) J. Biol. Chem. 269, 22853-22857). Direct comparisons between S. cerevisiae strain DTY167, harboring a deletion of the YCF1 gene, and the isogenic wild type strain, DTY165, demonstrate that YCF1 is required for increased resistance to the toxic effects of the exogenous glutathione S-conjugate precursor, 1-chloro-2,4-di-nitrobenzene, as well as cadmium. Whereas membrane vesicles isolated from DTY165 cells contain two major pathways for transport of the model compound S-(2,4-dinitrophenyl)glutathione (DNP-GS), an MgATP-dependent, uncoupler-insensitive pathway and an electrically driven pathway, the corresponding membrane fraction from DTY167 cells is more than 90% impaired for MgATP-dependent, uncoupler-insensitive DNP-GS transport. Of the two DNP-GS transport pathways identified, only the MgATP-dependent, uncoupler-insensive pathway is subject to inhibition by glutathione disulfide, vanadate, verapamil, and vinblastine. The capacity for MgATP-dependent, uncoupler-insensitive conjugate transport in vitro strictly copurifies with the acuolar membrane fraction. Intact DTY165 cells, but not DTY167 cells, mediate vacuolar accumulation of the quorescent glutathione-conjugate, monochlorobimane-GS. Introduction of plasmid borne, epitope-tagged gene encoding functional YCF1 into DTY167 cells alleviates the 1-chloro-2,4-dinitrobenzene-hypersensitive phenotype concomitant with restoration of the capacity of vacuolar membrane vesicles isolated from these cells for MgATP-dependent, uncoupler-insensitive DNP-GS transport. On the basis of these findings, the YCF1 gene of S. cerevisiae is inferred to encode an MgATP-energized, uncoupler-insensitive vacuolar glutathione S-conjugate transporter. The energy requirements, kinetics, substrate specificity, and inhibitor profile of YCF1-mediated transport demonstrate that the vacuolar glutathione conjugate pump of yeast bears a strong mechanistic resemblance to the MRP1-encoded transporter of mammalian cells and the cognate, but as yet molecularly undefined, function of plant cells.