In isolated rat pancreatic islets, glucose (5.6,11.1, and 16.7 mM) significantly increased reduced glutathione (GSH) and decreased oxidized glutathione (GSSG) levels in a dose-related manner. This was paralleled by a concomitant increase of NADPH and a decrease of NADP. The change of the GSH level occurred as quickly as one minute after addition of glucose. Exogenous insulin (200, 400, and 800 μU/ml) significantly decreased islet GSH levels in the presence of 5.6 and 16.7 mM glucose and significantly inhibited the insulin-releasing effect of the thiol reagent parachloromercuribenzoate (p-CMB) and tolbutamide. These data, together with earlier observations, suggest that GSH levels in pancreatic islets are increased by glucose and decreased by exogenous insulin via their effects on the pentose phosphate shunt and NADPH. Our results are compatible with the hypothesis that glucose and exogenous insulin, by modifying the redox state of the NADPH/NADP and GSH/GSSG systems, modulate the sensitivity of the β-cell to the insulin-triggering actions of glucose, p-CMB, and tolbutamide.