Glutathione synthesis, a vital cellular process, depends on L-cystine uptake by the amino acid transporter, System X−C. Here we show that a second transporter, System X−C, is required for normal System X−C activity and glutathione maintenance by employing somatic cell mutants of CHO-K1. Uptake by System X−Cin two X−C -null mutants is significantly lower than that of CHO-Kl, either under control conditions or after prolonged treatment with an electrophile. In addition, levels of glutathione in control and treated mutant cells are less than half those of wild-type CHO-K1 or of a pseudorevertant. The significance of this reduction was tested by chemical challenge: mutants are twofold more sensitive than wild type to reactive oxygen species generated by phenylbenzoquinone and to damage produced by the anticancer drug, cisplatin. These results suggest that System X−C provides a significant portion of the glutamate used to energize the uptake of cystine required for the synthesis of glutathione.