The synthesis of glutathione in Escherichia coli K 12 was studied in crude, cell-free extracts. The pH optima and the apparent Km values for the substrates have been determined for both synthesizing enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase. gamma-Glutamylcysteine synthetase was found to be approximately twice as active as glutathione synthetase. In a growing culture, the cellular level of GSH showed a considerable increase up to 6.6 mumol per ml cell pellet in the stationary growth phase. GSSG was not detectable. The levels of the enzymes remained constant, indicating that glutathione biosynthesis depends at least in the beginning on the availability of the component amino acids. The pathway is controlled by feedback inhibition and not by repression.