We have recently shown that mitoK_ATP opening is required for the inotropic response to ouabain. Because mitoK_ATP opening is also required for most forms of cardioprotection, we investigated whether exposure to ouabain was cardioprotective. We also began to map the signaling pathways linking ouabain binding to Na,K-ATPase to the opening of mitoK_ATP. In Langendorff-perfused rat hearts, 10-80 µM ouabain given before the onset of ischemia resulted in cardioprotection against ischemia-reperfusion injury, as documented by an improved recovery of contractile function and a reduction of infarct size. In skinned cardiac fibers, a ouabain-induced protection of mitochondrial outer membrane integrity, adenine nucleotide compartmentation and energy transfer efficiency was evidenced by a decreased release of cytochrome c and preserved K_1/2 (ADP) and ANT-miCK coupling, respectively. Ouabain-induced positive inotropy was dose-dependent over the range studied, whereas ouabain-induced cardioprotection was maximal at the lowest dose tested. When compared to bradykinin (BK)-induced preconditioning, ouabain was equally efficient. However, the two ligands clearly diverge in the intracellular steps leading to mitoK_ATP opening from their respective receptors. Thus, BK-induced cardioprotection was blocked by inhibitors of PKG or guanylyl cyclase (GC), whereas ouabain-induced protection was not blocked by either agent. Interestingly, however, ouabain-induced inotropy appears to require PKG and GC. Thus, 5-HD (a selective mitoK_ATP inhibitor), MPG (a reactive oxygen species scavenger), ODQ (a GC inhibitor), PP2 (a src kinase inhibitor), and KT 5823 (a Protein kinase G inhibitor) abolished preconditioning by bradykinin and blocked the inotropic response to ouabain. However, only PP2, 5-HD and MPG blocked ouabain-induced cardioprotection.