1. Numerous studies have demonstrated that ischaemic post-conditioning (IPoC) protects adult rats from myocardial ischaemia-reperfusion (I/R) injury. Recent evidence suggests compromised cardioprotection by IPoC in aged mice. The present study was designed to test the hypothesis that IPoC protects against I/R injury in aged hearts, potentially through a phosphatidylinositol 3-kinase (PI3-K)-Akt- and glycogen synthase kinase (GSK)-3beta-dependent mechanism. 2. Hearts from adult (3-4 months) or aged (16-18 months) Sprague-Dawley rats were subjected in vivo to 30 min ischaemia followed by 3 h reperfusion. Ischaemic post-conditioning (four cycles of 10 s reperfusion-10 s ischaemia) was applied at the beginning of reperfusion, either alone or in combination with the PI3-K inhibitor LY294002 (0.3 mg/kg). Infarct size and the phosphorylation of Akt and GSK-3beta were determined. 3. Ischaemic post-conditioning reduced infarct size in both adult and aged rat hearts. This protection was accompanied by a significant increase in phosphorylation of Akt and GSK-3beta. LY294002 abolished the IPoC-induced phosphorylation of Akt and GSK-3beta, as well as the infarct-limiting effect of IPoC in adult and aged rats. In addition, IPoC significantly attenuated plasma concentrations of creatine kinase and lactate dehydrogenase after reperfusion in both adult and aged rats. 4. In conclusion, IPoC, at the onset of reperfusion, reduces myocardial infarct size in both adult and aged rat hearts, potentially through a PI3-K-, Akt- and GSK-3beta-dependent mechanism.