The estrogen receptor (ER) mediates estrogenic activity in a variety of organs, including those in the reproductive, cardiovascular, immune, and central nervous systems. Experimental studies have demonstrated that 17β-estradiol (E2) protects the heart from ischemia-reperfusion injury. Two estrogen receptors, ERα and ERβ, mediate the actions of estrogen; however, it is not certain which ER mediates the cardioprotective effects of E2. In the present study, the ER-selective agonists 4,4′,4′′-[4-propyl-(1 H)-pyrazole-1,3,5-triyl]tris-phenol (PPT; ERα) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; ERβ) were assessed for their cardioprotective potential in an in vivo rabbit model of ischemia-reperfusion injury. Anesthetized female rabbits were administered PPT (3 mg/kg), DPN (3 mg/kg), E2 (20 μg/rabbit), or vehicle intravenously 30 min before a 30-min occlusion of the left anterior descending coronary artery followed by 4 h of reperfusion. Acute treatment with E2 (17.7 ± 2.9%; P < 0.001) and PPT (18.1 ± 2.9%; P < 0.001), but not DPN (45.3 ± 2.4%) significantly decreased infarct size as a percent of area at risk compared with vehicle (45.3 ± 2.4%). Coadministration of PPT or E2 with the ER antagonist ICI-182,780 limited the infarct size-sparing effect of the compounds (43.8 ± 6.6% and 40.6 ± 5.7% respectively, expressed as a percentage of risk region). PPT reduced the release of cardiac-specific troponin-I and reduced the tissue deposition of the membrane attack complex and C-reactive protein similar to that of E2. The results indicate that activation of ERα, but not ERβ, is required for the observed cardioprotective effects of E2.