—Influx of Ca²⁺ into myocytes via Na⁺/Ca²⁺ exchange may be stimulated by the high levels of intracellular Na⁺ and the changes in membrane potential known to occur during ischemia/reperfusion. This increased influx could, in turn, lead to Ca²⁺ overload and injury. Overexpression of the cardiac Na⁺/Ca²⁺ exchanger therefore may increase susceptibility to ischemia/reperfusion injury. To test this hypothesis, the hearts of male and female transgenic mice, overexpressing the Na⁺/Ca²⁺ exchange protein, and hearts of their wild-type littermates, were perfused with Krebs-Henseleit buffer and subjected to 20 minutes of ischemia and 40 minutes of reperfusion. Preischemic left ventricular developed pressures and +dP/dt_max, as well as −dP/dt_min, were higher in the male transgenic hearts compared with wild-type, implying a role for Na⁺/Ca²⁺ exchange in the contraction, as well as the relaxation, phases of the cardiac beat. Postischemic function was lower in male transgenic than in male wild-type hearts (7±2% versus 32±6% of preischemic function), but there was no difference between female transgenic and female wild-type hearts, both at ≈30% of preischemic function. To assess whether this male/female difference was due to female-specific hormones such as estrogen, the hearts of bilaterally ovariectomized and sham-operated transgenic females were subjected to the same protocol. The functional recoveries of ovariectomized female transgenic hearts were lower (17±3% of preischemic function) than those of wild-type and sham-operated transgenic females. The lower postischemic functional recovery in the male transgenic and female ovariectomized transgenic hearts correlated with lower recoveries of the energy metabolites, ATP and phosphocreatine, as measured by ³¹P nuclear magnetic resonance spectroscopy. Alternans were observed during reperfusion in male transgenic and female ovariectomized transgenic hearts only, consistent with intracellular Ca²⁺ overload. Western analyses showed that alterations in the expression of the Na⁺/Ca²⁺ exchange or L-type Ca²⁺ channel proteins were not responsible for the protection observed in the female transgenic hearts. In conclusion, in males, overexpression of the Na⁺/Ca²⁺ exchanger reduced postischemic recovery of both contractile function and energy metabolites, indicating that the Na⁺/Ca²⁺ exchanger may play a role in ischemia/reperfusion injury. From the studies of females, however, it appears that this exacerbation of ischemia/reperfusion injury by overexpression of the Na⁺/Ca²⁺ exchanger can be overcome partially by female-specific hormones such as estrogen.