Mitochondria are the major effectors of cardioprotection by procedures that open the mitochondrial ATP-sensitive potassium channel (mitoKATP), including ischemic and pharmacological preconditioning. MitoKATP opening leads to increased reactive oxygen species (ROS), which then activate a mitoKATP-associated PKCε, which phosphorylates mitoKATP and leaves it in a persistent open state (Costa, ADT and Garlid, KD. Am J Physiol 295, H874-82, 2008). Superoxide (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (HO•) have each been proposed as the signaling ROS but the identity of the ROS responsible for this feedback effect is not known. Superoxide was excluded in earlier work on the basis that it does not activate PKCε and does not induce mitoKATP opening.To further examine the identity of the signaling ROS, respiring rat heart mitochondria were preincubated with ATP and diazoxide to induce the phosphorylation-dependent open state, together with agents that may interrupt feedback activation of mitoKATP by ROS scavenging or by blocking ROS transformations. Swelling assays of the preincubated mitochondria revealed that dimethylsulfoxide (DMSO), dimethylformamide (DMF), deferoxamine, trolox, and bromoenol lactone (BEL) each blocked the ROS-dependent open state but catalase did not interfere with this step. The lack of a catalase effect and the inhibitory effects of agents acting downstream of HO• excludes H2O2 as the endogenous signaling ROS and focuses attention on HO•. In support of the hypothesis that HO• is required, we also found that HO•-scavenging by DMF blocked cardioprotection by both ischemic preconditioning and diazoxide in the Langendorff perfused rat heart. HO• itself cannot act as a signaling molecule, because its lifetime is too short and it reacts immediately with nearest neighbor phospholipids and proteins. Therefore, these findings point to a product of phospholipid peroxidation, such as hydroperoxy-fatty acids. Indeed, this hypothesis was supported by the finding that hydroperoxylinoleic acid (LAOOH) opens the ATP-inhibited mitoKATP in isolated mitochondria. This effect was blocked by the specific PKCε inhibitor peptide εV1-2, showing that LAOOH activates the mitoKATP-associated PKCε. During ischemia, catabolism of mitochondrial phospholipids is accelerated, causing accumulation of plasmalogens and free fatty acids (FA) in the heart by the action of calcium independent phospholipases A2 (iPLA2). We first assessed the role of FAs and hydroxy FAs on mitoKATP opening and cardioprotection. Swelling assays of isolated rat heart mitochondria showed that naturally formed free FAs inhibit mitoKATP opening and that they are more potent inhibitors of the pharmacological open state of mitoKATP than the phosphorylation-dependent open state. That is, sustained mitoKATP opening induced by the phosphorylation-dependent feedback loop is more resistant to FA inhibition than direct mitoKATP opening by a potassium channel opener. Moreover, rat hearts perfused with micromolar concentrations of FA were resistant to cardioprotection by diazoxide or ischemic preconditioning. Racemic bromoenol lactone (BEL), a selective inhibitor of iPLA2, confers protection to otherwise untreated Langendorff perfused hearts by preventing ischemic FA release. To bring this story full circle, BEL blocks protection afforded by preconditioning and postconditioning by preventing the iPLA2-mediated release of FAOOH generated in the conditioned heart. HO• resulting from mitoKATP opening oxidizes polyunsaturated fatty acid components of the membrane phospholipids, resulting in a peroxidized side chain. FAOOH must be released in order to act on the mitochondrial PKCε, and this is achieved by the action of iPLA2. iPLA2 is essential for most modes of cardioprotection because it catalyzes the release of FAOOH. This fully supports the hypothesis that the second messenger of cardioprotective ROS-mediated signaling is hydroperoxy fatty acid (FAOOH), a downstream oxidation product of HO•.