Cystine is a good acceptor of the gamma-glutamyl group of gamma-glutamyl donors in the reaction catalyzed by gamma-glutamyl transpeptidase. The product of the enzymatic reaction and an authentic sample of gamma-glutamylcystine were shown to exhibit identical chromatographic and electrophoretic behaviors; acid hydrolysis gave equimolar amounts of cystine and glutamate. In studies with two gamma-glutamyl donors, apparent Km values in the neighborhood of 0.3 mM were found for L-cystine; these values are not far from the concentrations of L-cystine in mammalian blood plasma. At an amino-acid acceptor concentration of about 0.5 mM, L-cystine is somewhat more active than L-glutamine, and much more active than L-cystein. L-gamma-Glutamyl-L-cystine was found to be a good substrate of gamma-glutamyl cyclotransferase. These observations thus indicate that L-cystine is a very active substrate of the gamma-glutamyl transpeptidase-gamma-glutamyl cyclotransferase pathway. In relation to the hypothesis that the gamma-glutamyl cycle functions in animo-acid transport, it may be significant that glutathione (which is the most abundant intracellular form) is a much better gamma-glutamyl donor than glutathione disulfide, while the predominant extracellular form-cystine-is a much better gamma-glutamyl acceptor substrate than cystein.