This study investigated the impact of testosterone on myocardial ischemia-reperfusion injury and corresponding intracellular calcium ([Ca2+]i) metabolism. Nonorchiectomized mature male Wistar rats were randomly assigned to placebo, a single dose of testosterone undecanoate, or 5alpha-dihydrotestosterone. In a further series, orchiectomized rats were treated with placebo. After 2 wk of treatment, the hearts were removed and placed in a Langendorff setup. The isolated, buffer-perfused hearts were subjected to 30 min of no-flow ischemia and 30 min of reperfusion. Recovery of myocardial function was measured by analyzing pre- and postischemic left ventricular (LV) systolic/diastolic pressure and coronary perfusion pressure simultaneously, together with [Ca2+]i handling (aequorin luminescence). Calcium regulatory proteins were analyzed by Western blotting. LV weight/body weight ratio was increased after administration of testosterone vs. orchectomized rats. The recovery of contractile function was improved in testosterone-treated rats: at the end of the reperfusion, LV systolic pressure was higher and end-diastolic pressure was lower in testosterone-treated rats. End-ischemic [Ca2+]i and [Ca2+]i overload upon reperfusion was significantly lower in testosterone vs. orchiectomized rats, too. However, levels of calcium regulatory proteins remained unaffected. In conclusion, administration of testosterone significantly improves recovery from global ischemia. These beneficial effects are associated with an attenuation of reperfusion induced [Ca2+]i overload.