The MRP (multidrug resistance protein) gene, a member of the ubiquitous superfamily of ATP-binding cassette transporters, is associated with the multidrug resistance of mammalian cells to natural product anticancer agents. We have previously shown that abrogation of MRP expression by gene targeting leads to hypersensitivity to several drugs. In two independently produced MRP double knockout clones, the baseline export of glutathione (GSH) was one-half that of wild-type embryonic stem (ES) cells. The export of GSH from wild-type ES cells, but not from the MRP double knockout clones, increased in the presence of etoposide (VP-16) and sodium arsenite, accompanied by equivalent decreases in intracellular levels of GSH. In the two MRP double knockout clones, the intracellular steady-state concentration of etoposide was twofold greater than that in wild-type cells. Depletion of intracellular GSH by d,l-buthionine sulfoximine increased the intracellular accumulation of radiolabeled etoposide in parental ES cells up to the level present in the two MRP knockout clones but did not change etoposide levels in the MRP knockout clones. These observations provide evidence that: (a) MRP exports GSH physiologically, presumably in association with an endogenous compound(s); (b) baseline MRP expression protects cells from the toxic effects of xenobiotics by effluxing the xenobiotics and GSH from the intracellular compartment into the extracellular medium by a co-transport mechanism; and (c) disruption of the gene encoding MRP abrogates the cotransport of xenobiotics and GSH.