Bradykinin (BK) triggers preconditioning via a pathway which include B2 receptors, nitric oxide (NO), protein kinase G (PKG), mitochondrial KATP channels (mKATP) and reactive oxygen species (ROS). Postconditioning (PostC) allows post-ischemic accumulation of autacoids, which trigger protection. We tested if PostC-triggering includes BK and its downstream pathway. Two-hundred rats were anesthetized with ketamin and decapitated, then isolated hearts underwent 30 min ischemia and 120 min reperfusion. Infarct size was evaluated using nitro-blue-tetrazolium staining. Data are mean±SEM. One-Way ANOVA and post-test Tukey’s HSD were used for statistical analysis. In Control hearts infarct size was 61±5% of risk area. PostC (5 cycles of 10 s reperfusion/ischemia) reduced infarct size to 22±4% (p<0.01). The infarct-sparing effect of PostC was abolished by the following antagonists given for 3 min only, while performing PostC maneuvers: a) the B2 BK receptor-antagonists HOE140 (100 nM) or WIN64338 (100 μM); b) the NO synthase-inhibitor L-nitro-arginine-methylester (100µM); c) the PKG-blocker 8-bromoguanosine-3',5'-cyclic-monophosphorothioate (1 µM); d) mKATP blocker 5-hydroxydecanoate (100 μM). Since 3 min of BK-infusion (100nM) did not reproduce PostC-protection, protocols with Intermittent-BK infusion were used to mimic PostC: a) 5 cycles of 10 s oxygenated-no-BK/oxygenated+BK buffer; b) 5 cycles of 10 s oxygenated-no-BK/hypoxic+BK buffer. Both protocols with Intermittent-BK attenuated infarct size (36±5% and 38±4%, respectively; p<0.05 vs Control and NS vs PostC for both; NS vs each other). Intermittent-BK protection was abolished by the same antagonists used to prevent PostC-protection. Intermittence of re-oxygenation only (5 cycles of 10 s oxygenated/hypoxic buffer) did not reproduce PostC. Yet, cardioprotection was triggered by intermittent mKATP activation with diazoxide (30μM), but not by intermittent ROS generation with purine/xanthine oxidase. However, ROS scavengers [N-acetyl-L-cysteine (4 mM) or 2-mercaptopropionylglycine (300 μM)], given for 3 min only, abolished either PostC-, Intermittent BK- or diazoxide-induced protection. Intermittent targeting of specific cellular sites (i.e. BK B2 receptors and mKATP channels) during early reperfusion triggers PostC-protection via a ROS signaling. Since neither intermittent oxygenation nor exogenous ROS generators can trigger protection, it is likely that metabolites are washed-out and that exogenous ROS do not reach the right compartment. Therefore, intermittent autacoid accumulation and ROS compartmentalization may play a pivotal role in PostC-triggering.