Abstract The aim of this study was to define dietary conditions liable to elevate the circulating and tissue levels of glutathione (GSH). For this purpose, GSH synthesis and availability have been compared after supplementation of a low protein diet (10% casein) with 0.6% methionine or after adaptation to a high protein diet (30% casein), the final dietary methionine levels were similar in both cases. To compare the effects of an acute addition of sulfur amino acids to the diet with long-term adaptative changes on GSH status, rats adapted to the 10% casein diet received as a final meal (i) the diet supplemented with 0.6% methionine or (ii) a 30% casein diet. With a 10% casein diet, the plasma and tissue concentrations of methionine and cysteine seem to constitute limiting factors for GSH synthesis. However, in animals adapted to a high protein diet or to a diet supplemented in methionine, the hepatic GSH content was quite enhanced (respectively 1.9 and 2.6 fold that measured in control diet), in keeping with the marked rise of the hepatic cysteine (respectively 2 and 5 fold that measured in the control diet). Acute administration of a final 30% casein meal or 10% casein meal supplemented in methionine induced greater changes in the hepatic GSH content than observed in long-term experiments. Compared with the liver, the muscle and heart GSH content was poorly affected by changes in sulfur amino acids availability or nutritional state. The liver appears as a net site of GSH release, the magnitude of this release was not entirely proportional to the hepatic pool, particularly during the postabsorptive period. Moreover, during this period the hepatic GSH content was markedly depressed compared with the fed state, whatever the dietary conditions. With diets providing a high availability in sulfur amino acids, the activity of the liver γ-glutamyl cysteine synthetase was markedly depressed, even in acute experiments. In vitro experiments confirmed in vivo results, and showed that until its extracellular concentration reached 0.4 mM, methionine may constitute a limiting factor for GSH synthesis in isolated hepatocytes.