In order to study the cell signaling events leading to 17-estradiol (E2)-induced acute cardioprotection, isolated rat hearts were subjected to three 5 min cycles of 10 µM E2 prior to 30 min of regional ischemia, followed by 2 hrs of reperfusion. Protection was judged by changes in infarct size in % of risk zone volume. To test the importance of phosphoinositide 3-kinase (PI3-K), protein kinase C (PKC) or reactive oxygen species (ROS) in E2-induced protection, wortmannin (1 µM), chelerythrine (2 µM) and 2-mercaptopropionylglycine (300 µM) respectively were combined with E2 exposure. Changes in phosphorylation of protein kinase B (PKB) and selected PKC isoforms were tested by immunoblotting of total lysates and subcellular fractions, along with assessment of PKC translocation from soluble to membrane fraction of heart tissue homogenates. Intracellular ROS levels induced by E2 preconditioning were investigated. E2 preconditioning led to significant reduction in infarct size from 31.8±5.3 to 20.2±2.6% in male and from 42.7±4.7 to 17.1±3.4% in female hearts (p<0.05). Protection was abolished by wortmannin (30.0±3.2%), chelerythrine (45.1±4.4%) and 2-mercaptopropionylglycine (36.8±4.7%). E2 preconditioning induced phosphorylation of PKB, PKCs and and membrane translocation of PKCs and . Intracellular ROS levels were found elevated after transient treatment with hormone. Therefore, our data demonstrate the ability of E2 to induce preconditioning-like cardioprotection via cell signaling events shared by classical ischemic preconditioning.