Adenosine is a powerful trigger of ischemic preconditioning (IPC). Myocardial ischemia induces intracellular ATP degradation to adenosine which is subsequently released and fuels interstitial adenosine. Extracellular adenosine formation by ecto-5'-nucleotidase (CD73) presumably contributes only little in this process. A recent report of a lack of IPC in CD73-deficient (CD73^-/-) mice (Eckle, Circ. 115: 1581, 2007) was thus surprising. We demonstrated in vitro similar venous adenosine concentrations following ischemia and identical IPC in wild type (WT) and CD73^-/- female hearts (Acta Physiol. 2009). To extend and confirm these data in vivo, we established an open-chest model of IPC and myocardial infarction and compared the effect of IPC in male and female, WT and CD73^-/- mice. A total of 50 C57/BL6 mice were anesthetized with pentobarbital-Na and mechanically ventilated. Four cycles of 5 min ischemia and 5 min reperfusion to induce IPC were exerted before an index ischemia of 45 min. Following 90 min of reperfusion, hearts were cut into 5 slices and infarct size and the area at risk were determined by TTC and Evans Blue staining. In absence of IPC, the index ischemia induced an infarct size of 45,3±8,9 resp. 40,5±8 % of area at risk (WT, n=12 resp. CD73^-/-, n=13), which was identical in both groups as well as in male and female hearts (n=5–7, resp.). IPC induced an impressive cardioprotection, alleviating infarct size by >40%. This was true both in WT and CD73^-/- hearts: Infarct size was reduced to 26,3±8 resp. 22,6±6,6 % of area at risk (n=12 resp. 13). The extent of IPC-induced cardioprotection in male and female mice (n=5–7, resp.) was again identical. In conclusion, consistent with a dominant intracellular adenosine formation in ischemia, but contrary to a previous report, the beneficial effects of IPC in the heart are not affected by the genetic ablation of CD73. Thus, the extracellular adenosine formation is unlikely to contribute to adenosine's well known cardioprotective effect.