We have previously demonstrated the effects of estrogen on modulation of myocardial ATP-sensitive K+(KATP) channel. Previous studies have demonstrated that activation of mitochondrial KATPchannel is a major contributor of ischemic cardioprotection. The purpose of the present study was to investigate the role of KATPchannel in estrogen-induced myocardial protection after ischemia/reperfusion in dogs. Anaesthetized dogs were subjected to 60 min of left anterior descending coronary artery occlusion followed by 2 h of reperfusion. In a first study to characterize effects of sex and the dose-response profile of estrogen on infarct size, the drug was intravenously administered at 10 or 20 μ g/kg. In a second study to investigate the cardioprotective mechanisms of estrogen, vehicle, preconditioning or 17 β -estradiol (10 μ g/kg) was given, beginning 15 min prior to the 60 min occlusion period in the presence or absence of 5-hydroxydecanoate (5-HD). In the first study, administration of 17 β -estradiol resulted in a significant, dose-dependent limitation of infarct size. Estrogen administration provided myocardial protection of similar magnitude in both males and females. In the second study, infarct size in control animals averaged 39±5% of the risk region, compared with 14±5% of the risk region in estrogen-treated dogs and 6±5% of the risk region in preconditioning dogs (both P<0.0001 v controls). Pretreatment with 5-HD completely abolished preconditioning- and estrogen-induced cardioprotection. Estrogen limits myocardial infarction size resulting from coronary artery occlusion and reperfusion in a dose-dependent fashion, irrespective of gender difference. The infarct size-limiting effect of estroge was abolished by 5-HD, suggesting that the cardioprotective effect of estrogen may result from activation of myocardial mitochondrial KATPchannels.