Glutathione synthesis, a vital cellular process, depends on L-cystine uptake by the amino acid transporter, System XC. Here we show that a second transporter, System XC, is required for normal System XC activity and glutathione maintenance by employing somatic cell mutants of CHO-K1. Uptake by System XC in two XC -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 XC provides a significant portion of the glutamate used to energize the uptake of cystine required for the synthesis of glutathione.