Motivation: We previously showed a protective role for the intermittent dyssynchrony (pacing postconditioning (PPC)), induced by ventricular pacing immediately at the beginning of reperfusion. The aim of this study was to determine possible gender differences and the effects of estrogen (E2) on ischemia reperfusion injury (I/R) and the outcome of PPC. Methods: Hearts isolated from Wistar rats anesthetized with intraperitoneal injection of sodium pentobarbital (60 mg/kg) and anticoagulated with heparin (1000U/Kg body weight) through the femoral vein were used for this study. The hearts (n=8 per group) were canulated to a modified Langendorff system. Controls were subjected to 30 minutes unprotected ischemia. PPC protocol was 3 cycles of 30 seconds left ventricular (LV) pacing alternated with 30 seconds right atrial (RA) pacing at the beginning of reperfusion. To study the short-term effect of E2, the hearts were perfused with 0.7 ng/ml E2 for 15 minutes at the beginning of reperfusion. For E2 long-term treatment female rats were anesthetized with intraperitoneal injection of sodium pentobarbital (60 mg/kg), overiectomized and substituted with 60 days E2 or placebo releasing pellets. Hearts isolated from these animals were subjected to ischemia and PPC. All groups were allowed a period of 30 minutes reperfusion. Hemodynamics were computed by a data acquisition program. Infarct size was determined by triphenyltetrazolium chloride (TTC) staining. Results: Unprotected I/R resulted in poor recovery of hemodynamics (left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (Pmax), Maximal derivative of left ventricular pressure (dP/dtmax), coronary flow (CF), and coronary vascular resistance (CVR)) compared to baseline data (p<0.02). PPC significantly decrease (P<0.05) the infarct size and improved hemodynamics in the male and female hearts compared to controls. Both short- and long-term E2 treatments did not protect the heart against I/R injury. However, PPC did not show any protection in hearts treated with E2 for 6 weeks. Significant (P<0.003) decrease in infarct size and improvement in hemodynamics were seen in 6 weeks placebo treated hearts compared to E2 treated hearts and controls. Conclusions: There were no gender differences in PPC protection. However Long-term E2 treatments abrogated PPC protection, short-term E2 treatment did not protect the heart.