The aim of this study was to assess early preconditioning protection against stunning in conscious sheep and analyze the role of ATP-dependent potassium (KATP) channels in the protective mechanism. Chronically instrumented animals were submitted to a 12 min reversible ischemia and 2 h reperfusion. Early preconditioning, consisting of six 5 min occlusion-5 min reperfusion periods, followed by 45 min normoperfusion before the prolonged ischemia protected against stunning (P < 0.01). In these experimental conditions, current agents used to analyze sarcolemmal (sKATP) and mitochondrial (mKATP) KATP channels could not clearly establish their participation in the protective mechanism. At doses that inhibit sKATP channels they were unable to block preconditioning protection against stunning (glibenclamide) or conversely, blocked preconditioning at doses that do not inhibit these channels (HMR1098). Moreover, both mKATP channel agonists (diazoxide) and antagonists (5HD) protected against stunning, a response that could be due to their effect via an alternative mitochondrial pathway.