The metabolism of glutathione and activities of its related enzymes were investigated in erythrocytes from patients with diabetes mellitus. A decrease in the levels of the reduced form of glutathione and an increase in the levels of glutathione disulfide were observed in erythrocytes from diabetics whose fasting plasma glucose was more than 140 mg/dl. The activity of glutathione reductase decreased in diabetics, while that of glutathione peroxidase did no change. ATP-depended outward transport of glutathione disulfide also decreased in diabetics. These data suggest that the increase in the levels of glutathione disulfide in erythrocyte from diabetics is brought about by the decreased transport activity of glutathione disulfide through the erythrocyte membrane together with a decrease in the activity of glutathione reductase. The activity of gamma-glutamylcysteine synthetase was significantly lower in diabetics than in normal controls. Glycated gamma-glutamylcysteine synthetase determined using a boronate affinity column chromatography was higher in diabetics than in normal controls. The rate of glutathione synthesis using (H3)-glycine decreased in diabetics. The decrease is the levels of reduced form of glutathione is erythrocytes of diabetics is thought to be brought about by impaired glutathione synthesis. In order to study the mechanism by which glutathione synthesis is impaired, gamma-glutamylcysteine synthetase was purified from human erythrocytes. The molecular weight of the purified enzyme was 60K. A single band was observed on SDS polyacrylamide gel electrophoresis. When the purified enzyme was incubated with glucose, the enzyme activity decreased dependent on the incubation time. These data suggest that the impaired glutathione synthesis in diabetics is brought by glycation of gamma-glutamylcysteine synthetase. As conclusion, glutathione metabolism is impaired in erythrocytes from diabetics which weaken the defence mechanism against oxidative stress in these patients.