We previously showed that chronic hypoxia decreases activity of intestinal glutathione (GSH)-dependent enzymes and is associated with a proximal-to-distal gradient of enzyme activity, suggesting reduced detoxication capacity in ileal cells. To assess whether hypoxia affects metabolism of hydroperoxides, jejunal and ileal enterocytes from rats exposed to air (n = 11) or 10% O2 (n = 9) for 10 days were exposed to 300 microM tert-butyl hydroperoxide (t-BH). The initial rate of hydroperoxide metabolism was 50-90% faster in hypoxic enterocytes, but cell killing was enhanced. Metabolism in normoxic, but not hypoxic, cells was enhanced threefold by addition of 10 mM glucose. Hypoxic enterocytes exhibited a higher baseline GSH/GSH disulfide (GSSG) ratio but a larger percent decrease after t-BH exposure. t-BH induced a 35-40% decrease in protein-bound sulfhydryl groups in normoxic and hypoxic enterocytes, but protein-bound sulfhydryl was protected by glucose in normoxic cells only. Metabolic response to substrate load or hydroperoxide challenge was assessed by measurement of cellular O2 consumption. Hypoxia, but not normoxia, increases and decreases O2 consumption on exposure to glucose and oxidant, respectively, suggesting metabolic dysregulation. In summary, prolonged O2 deficiency induces loss of intestinal metabolic integrity that is associated with altered peroxide detoxication activity and mitochondrial respiratory function.