Perfusion of the heart with bradykinin triggers cellular signaling events that ultimately cause opening of mitochondrial ATP-sensitive K⁺-channels (mitoK_ATP), increased H₂O₂ production, inhibition of the mitochondrial permeability transition (MPT), and cardioprotection. We hypothesize that bradykinin interaction with its receptor induces the assembly of a caveolar signaling platform (signalosome) that contains the enzymes of the signaling pathway and that migrates to mitochondria to induce mitoKATP opening. We developed a novel method for isolating and purifying signalosomes from Langendorff-perfused rat hearts treated with bradykinin. Fractions containing the signalosomes were found to open mitoK_ATP in mitochondria isolated from untreated hearts via activation of a mitochondrial protein kinase C epsilon. MitoK_ATP opening required signalosome-dependent phosphorylation of an outer membrane protein. Immunodetection analysis revealed the presence of the bradykinin B2 receptor only in the fraction isolated from bradykinin-treated hearts. Immunodetection and immunogold labeling of caveolin-3, as well as sensitivity to cholesterol depletion and resistance to Triton X-100, attested to the caveolar nature of the signalosomes. Ischemic preconditioning, ischemic postconditioning and perfusion with ouabain, also led to active signalosome fractions that opened mitoK_ATP in mitochondria from untreated hearts. These results provide initial support for a novel mechanism for signal transmission from plasma membrane receptor to mitoK_ATP.