Gamma-Glutamyl-cysteine synthetase is inhibited by glutathione under conditions similar to those which prevail in vivo, thus strongly suggesting a physiologically significant feedback mechanism. Inhibition by glutathione, which is not allosteric, appears to involve the binding of glutathione to the glutamate site of the enzyme as well as to another enzyme site; the latter binding appears to require a sulfhydryl group since ophthalmic acid (gamma-glutamyl-alpha-aminobutyryl-glycine) is only a weak inhibitor. The finding that glutathione regulates its own synthesis by inhibiting synthesis of gamma-glutamyl-cysteine appears to explain observations on patients with 5-oxoprolinuria, who were shown to have a block in the gamma-glutamyl cycle consisting of a marked deficiency of glutathione synthetase and consequently of glutathione. These patients produce greater than normal amounts of gamma-glutamyl-cysteine, which is converted by the action of gamma-glutamyl cyclotransferase to 5-oxoproline; production of the latter compound exceeds the capacity of 5-oxoprolinase to convert it to glutamate. The apparent Km value for L-cysteine for gamma-glutamyl-cysteine synthetase (0.35 mM) is not far from intracellular concentrations of L-cysteine suggesting that the availability of L-cysteine may also play a role in the regulation of glutathione synthesis.