AIMS: Pre-treatment with mineralocorticoid receptor (MR) antagonists is reported to reduce myocardial infarct size from ischaemia/reperfusion. Here, we tested whether the MR antagonists potassium canrenoate and eplerenone could protect in the more clinically relevant schedule of administration at the end of ischaemia. METHODS AND RESULTS: In all models, hearts were subjected to 30 min regional ischaemia followed by 120 min (rabbits 4 h) reperfusion. A bolus of canrenoate 5 min prior to reperfusion in open-chest mice decreased infarct size in a dose-dependent manner. Maximum protection was seen at 1 mg/kg where infarction was 18% of that in the control (P < 0.001). Ecto-5'-nucleotidase (CD73) as well as adenosine A(2b) receptor knock-out mice could no longer be protected, suggesting a role for adenosine and the A(2b) receptor in the mechanism. A 1 mg/kg bolus of canrenoate prior reperfusion also reduced infarct size in open-chest rabbits. To explore the underlying mechanisms, we studied isolated rat hearts. Eplerenone (10 microM) at the end of ischaemia was similarly protective in the rat heart and the protection was abolished by co-treatment with inhibitors of the adenosine receptor, protein kinase C, PI3-kinase, and ERK. In addition, eplerenone or canrenoate treatment increased phosphorylation of the pro-survival kinases Akt and ERK1/2 at reperfusion in the rat hearts. CONCLUSION: Taken together, MR antagonists when given at the end of ischaemia are highly effective and potent cardioprotective drugs with a signalling similar to that of ischaemic pre-conditioning and, hence, could be a very promising candidate for the treatment of acute myocardial infarction in man.