The transport of L-cystine into cells of the mammalian brain is an essential step in the supply of cysteine for synthesis of the antioxidant glutathione. Uptake of L-cystine in rat brain synaptosomes occurs by three mechanisms that are distinguishable on the basis of their ionic dependence, kinetics of transport and specificity of inhibitors. Almost 90% of L-cystine transport is by a low-affinity, sodium-dependent mechanism (K(m)=473+/-146 microM), that is mediated by the X(AG)- family of glutamate transporters. Both L-glutamate (IC(50)=9.1+/-0.4 microM) and L-cysteine sulphinate (IC(50)=16.4+/-3.6 microM) are non-competitive inhibitors of sodium-dependent L-[(14)C]cystine transport, whereas L-trans-pyrrolidine-2,4-dicarboxylic acid (IC(50)=5.6+/-2.0 microM), L-serine-O-sulphate (IC(50)=13.2+/-5.4 microM), kainate (IC(50)=215+/-78 microM) and L-cysteine (IC(50)=363+/-63 microM) are competitive inhibitors. L-Cystine has no effect on the sodium-dependent uptake of D-[(3)H]aspartate. These results suggest that L-cystine binds to a site that is different from the L-glutamate recognition site on X(AG)- glutamate transporters. In rat brain slices, sodium-dependent transport of both L-glutamate and L-cystine is necessary for maintaining glutathione levels. Uptake of L-cystine is sensitive to inhibition by an increased extracellular concentration of L-glutamate, which has important implications for understanding conditions that may initiate oxidative stress.