We previously showed that the stimulation of heme-oxygenase 1 expression by high glucose and hydrogen peroxide (H₂O₂) in cultured rat islets is prevented by antioxidants and suggested that this effect of high glucose results from an oxidative stress. However, the role of oxidative stress in high glucose-induced -cell dysfunction is unclear. We therefore compared the preventative effects of N-acetyl-L-cystein (NAC), a free radical scavenger, and manganese(III)tetrakis (4-benzoic acid)porphyrin (MnTBAP), a superoxide dismutase/catalase mimetic agent, on the alteration of stimulus-secretion coupling induced in rat islets by overnight exposure to hydrogen peroxide (H₂O₂-treated islets) or one-week culture in 30 vs 10 mmol/l glucose (High-glucose vs Control islets). The features of -cell dysfunction differed between the two groups : reduced glucose-induced insulin secretion without changes in glucose sensitivity in H₂O₂-treated islets ; increased sensitivity to glucose with parallel reductions in insulin content and maximal rate of glucose-induced insulin secretion in High-glucose islets. The latter alterations were accompanied by a decrease in preproinsulin without changes in PDX1 mRNA levels. The functional alterations induced by H₂O₂ were significantly prevented by addition of NAC or MnTBAP in the culture medium. In contrast, neither NAC nor MnTBAP affected the functional alterations induced by high glucose. These results suggest that -cell dysfunction induced by 1 week culture in high glucose does not result from an increase in oxidative stress.