The role of glutathione (GSH) in myocardial antioxidant defense was investigated in Swiss-Webster mice either performing swim exercise to exhaustion or rested in both the GSH adequate (GSH-A) and GSH deficient (GSH-D) states. GSH deficiency was accomplished by injecting mice with L-buthionine [S,R]sulfoximine (BSO; 2 nmol/kg body wt, i.p.) and providing BSO (20 mM) in drinking water for 12 days. GSH and glutathione disulfide (GSSG) contents in the GSH-D hearts were decreased to 10 and 8%, respectively, of those in the GSH-A mice. This decrease was associated with a significant decline of the total glutathione level in the liver, skeletal muscle and plasma. Myocardial GSH peroxidase and GSH sulfur-transferase activities decreased significantly following GSH deficiency, whereas superoxide dismutase activity was significantly elevated. GSH deficiency did not affect exercise endurance performance. However, exhaustive exercise decreased GSH content in the myocardium of the GSH-A and GSH-D mice by 22 and 44% (p < 0.05), respectively. The GSH:GSSG ratio was not altered significantly following exercise because of a concomitant decrease in GSSG (p < 0.05). gamma-Glutamyltranspeptidase activity was significantly increased after exercise, especially in the GSH-D hearts (72%; p < 0.05). GSH content after exercise correlated negatively with exercise time in both GSH-A and GSH-D mice (p < 0.05). These data indicate that GSH is actively used in the myocardium during prolonged exercise at moderate intensity and that GSH deficiency is tolerated by the heart, possibly compensated for by an increased GSH uptake from the plasma.