Sleep deprivation in humans is widely believed to impair health, and sleep is thought to have powerful restorative properties. The specific physical and biochemical factors and processes mediating these outcomes however are poorly elucidated. Sleep deprivation in the animal model produces a condition that eventually becomes highly lethal, lacks specific localization, and is reversible with sleep, implying mediation by a biochemical abnormality. Metabolic and immunologic consequences of sleep deprivation point to a high potential for antioxidant imbalance. The objective therefore was to study glutathione content in the liver, heart, and lung, because glutathione is considered a major free radical scavenger that reflects the degree to which a tissue has been oxidatively challenged. We also investigated major enzymatic antioxidants including catalase and glutathione peroxidase, as well as indices of glutathione recycling. Catalase activity and glutathione content, which normally are tightly regulated, were both decreased in liver by 23 to 36 percent by 5 and 10 days of sleep deprivation. Such levels are associated with impaired health in other animal models of oxidative stress-associated disease. The decreases were accompanied by markers of generalized cell injury, and absence of responses by the other enzymatic antioxidants under study. Enzymatic activities in the heart indicated an increased rate of oxidative pentose phosphate pathway activity during sleep deprivation. Recovery sleep normalized antioxidant content in liver and enhanced enzymatic antioxidant activities in both the liver and the heart. The present results link uncompensated oxidative stress to health effects induced by sleep deprivation and provide evidence that restoration of antioxidant balance is a property of recovery sleep.