Formation of oxygen-derived free radicals and activation of the glutathione (GSH) redox cycle has been associated with impaired rat diaphragm performance. Diethylmaleate (DEM) given intraperitoneally irreversibly conjugates with GSH, resulting in marked decreases in tissue concentrations of GSH. We have investigated the effects of acute GSH depletion by DEM on diaphragmatic function during resistive breathing (RB) in the rat. The experimental groups were 1) control, 2) DEM alone, 3) RB, and 4) DEM with RB (DEM + RB). RB was obtained by inspiratory RB until the rats were unable to sustain 70% of maximum airway opening pressure. A portion of the diaphragm was frozen for biochemical assays, and the rest of the diaphragm was prepared for measurement of in vitro contractile properties, including maximum tetanic tension, twitch tension, force-frequency curves, and contraction times. DEM treatment produced a profound depletion of GSH in the DEM and DEM + RB groups. Neither DEM nor RB alone significantly altered diaphragm contractile properties. In DEM + RB rats, however, there was a significant decrease in maximum tetanic tension, twitch tension, and tetanic tension. These data reveal that DEM produced an acute depletion of GSH in the diaphragm without impairment of the muscle in nonstressed rats. In the presence of DEM-induced GSH depletion, RB did result in marked diaphragm impairment. The depletion of GSH and the subsequent impairment in diaphragm contractility after RB suggest that GSH may play an important role in protecting the diaphragm against oxidative stress associated with RB.