Chronic endogenous testosterone exposure adversely affects proinflammatory and proapoptotic signaling after ischemia reperfusion; however, it remains unknown whether a single acute testosterone exposure is equally detrimental. We hypothesized that acute exogenous testosterone infusion prior to ischemia would worsen myocardial functional recovery, increase the activation of mitogen activated protein kinases and caspase-3, and increase myocardial proinflammatory cytokine production. To study this, isolated-perfused rat hearts (Langendorff) from adult females and castrated males were subjected to 25 min ischemia and 40 min reperfusion with and without acute testosterone infusion (17beta-hydroxy-4-androstenone, 10ng/ml/min) prior to ischemia. Myocardial contractile function was continuously recorded. After ischemia/reperfusion, hearts were assessed for levels of testosterone (ELISA), expression of proinflammatory cytokines (ELISA), and activation of mitogen activated protein kinases and caspase-3 (Western blot). Data were analyzed with two-way ANOVA or student's t-test, p<0.05 statistically significant. All indices of post-ischemic functional recovery were decreased with acute exogenous testosterone compared to the untreated groups. Acute testosterone infusion increased activation of mitogen activated protein kinases and caspase-3 following ischemia / reperfusion. However, there were no significant differences in the myocardial proinflammatory cytokine production after brief testosterone infusion. A single acute exposure to exogenous testosterone prior to ischemia worsens myocardial functional recovery, and increases activation of mitogen activated protein kinases and caspase-3. These findings confirm the deleterious effects of testosterone on myocardium, elucidate the nongenomic mechanistic pathways of testosterone, and may have important clinical implications for patients who have acute exposure to exogenous testosterone.