The study investigated whether estradiol can prevent release of cytochrome c from mitochondria and induction of apoptosis after 30 and 60 min stop-flow heart ischemia in Langendorff-perfused female rat hearts. Pre-perfusion of hearts with 100 nM 17beta-estradiol prevented the loss of cytochrome c from mitochondria, its accumulation in cytosol, and inhibition of respiration during ischemia. Estradiol strongly reduced activation of caspase-3-like activity and decreased DNA strand breaks in the nuclei of cardiomyocytes (measured by TUNEL staining). The results show that 17beta-estradiol prevents the ischemia-induced release of cytochrome c from mitochondria, subsequent inhibition of mitochondrial respiration, and inhibits caspase activation and apoptosis. Therefore, inhibition of the intrinsic, mitochondria-mediated apoptotic pathway may be one of the mechanisms by which estrogens protect the heart against ischemic damage.