System x c – is one of amino acid transporters expressed in the plasma membrane of mammalian cells and specifically exchanges anionic forms of cystine and glutamate. This transporter plays an important role in maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of System x c -, xCT, is strongly induced by various stimuli, including oxidative stress. Constitutive expression of xCT is observed only in specific regions of the brain and immune tissues such as thymus and spleen. We generated xCT deficient mice and performed metabolite analysis of various tissues using capillary electrophoresis time-of -fight mass spectrometry, and found that no cystathionine was detected in thymus and spleen of xCT-deficient mice, although a significant amount of cystathionine was detected in these tissues in wild-type mice. These results suggest that cystathionine may be a physiological substrate of System x c -. Embryonic fibroblasts derived from wild-type mice did not survive more than 24 h in cystine-free medium, due to a marked decrease in intracellular glutathione levels. However, these cells survived and proliferated when incubated in cystine-free medium containing 0.1 mM cystathionine. In contrast, cells derived from xCT-deficient mice did not survive in the cystine-free medium, even when 0.1 mM cystathionine was added to the medium. Cystine uptake in the wild-type cells was significantly inhibited by cystathionine. When wild-type cells were incubated in the 0.1 mM cystathionine-containing buffer, release of intracellular glutamate was enhanced, and cystathionine concomitantly accumulated in the cells. To induce experimental hypercystathionemia, xCT-deficient and wild-type male mice (8-9 weeks) were injected with propargylglycine, a cystathionine γ-lyase inactivator, diluted in saline (ip, 50 mg/kg) daily for 3 days, and cystathionine in plasma, liver and thymus were measured. In plasma and liver of both xCT-deficient and wild-type mice, cystathionine was significantly increased. In the thymus of wild-type mice, cystathionine was substantially increased while no cystathionine was detected in the thymus of xCT-deficient mice. From these results, we have demonstrated that cystathionine functions as a physiological substrate of System x c – and is exclusively transported via System x c – in an exchange for glutamate in thymus.