The effects of hypoxia on glutathione (GSH) concentration and GSH-related enzyme and transport systems were studied in the small intestine of rats exposed to 8-10 days of 10.5% O2. Exposure to hypoxia resulted in a 40% lower GSH concentration in enterocytes and a 50% lower concentration in blood plasma. Activities of GSH-related detoxication enzymes in the intestinal epithelium were largely unaffected by hypoxic exposure. GSH degradation and synthesis rates in enterocytes isolated from hypoxic rats were comparable with rates in normoxic controls, but GSH uptake rate was decreased by 30%. Stimulation of absorption of GSH by phenylephrine, such as occurs in control rats, was not detectable in isolated, vascularly perfused intestines of hypoxic rats. Decreased GSH uptake was associated with enhanced transepithelial appearance of thiobarbituric acid-reactive substances in everted intestinal sacs incubated with peroxidized methyl linoleate. These results suggest that chronic hypoxia results in impaired uptake of GSH in the small intestine, and this may result in impaired GSH-related defense mechanisms in the small intestine.