Saccharomyces cerevisiae has two independent transport systems for the removal of arsenite from the cytosol. Acr3p is a plasma membrane transporter that confers resistance to arsenite, presumably by arsenite extrusion from the cells. Ycf1p, a member of the ABC transporter superfamily, catalyzes the ATP-driven uptake of As(III) into the vacuole, also producing resistance to arsenite. Vacuolar accumulation requires a reductant such as glutathione, suggesting that the substrate is the glutathione conjugate, As(GS)3. Disruption of either the ACR3 or YCF1 gene results in sensitivity to arsenite and disruption of both genes produces additive hypersensitivity. Thus, Acr3p and Ycf1p represent separate pathways for the detoxification of arsenite in yeast.