—The critical time for opening mitochondrial (mito) K_ATP channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29±3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 μmol/L diazoxide, a mito K_ATP channel opener, reduced infarction to 3±1% and 8±1%, respectively. The mito K_ATP channel closer 5-hydroxydecanoate (200 μmol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24±3 and 28±6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 μmol/L), another K_ATP channel closer, blocked the protection by PC only when administered early. These data suggest that K_ATP channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8±3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 μmol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 μmol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with N-(2-mercaptopropionyl) glycine (300 μmol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 μmol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K_ATP channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.