We studied the effect of 2-week treatment with estradiol 17β on myocardial glutathione concentration in dogs and isolated perfused rat heart subjected to brief coronary ischemia and reperfusion. Estradiol protected against ischemia/reperfusion-induced myocardial systolic shortening and malonylaldehyde production and increased myocardial glutathione concentration and glucose-6-phosphate dehydrogenase enzyme activity. Reduction of myocardial glutathione with buthionine sulfoximine to levels seen in the absence of estrogen reversed the protective effect of estradiol against myocardial dysfunction and lipid peroxidation associated with ischemia/reperfusion. These results suggest that the antioxidant effect of estradiol in ischemia/reperfusion may be mediated by regulation of myocardial glutathione metabolism.