We previously reported that pharmacological preconditioning of rabbit hearts with acetylcholine involves activation of phosphatidylinositol 3-kinase (PI3-K) through transactivation of the epidermal growth factor receptor (EGFR). Transactivation is thought to be initiated by cleavage of membrane-bound pro-heparin-binding EGF-like growth factor (HB-EGF) by a membrane metalloproteinase thus releasing HB-EGF which binds to the EGFR. This pathway leads to redox signaling with the generation of reactive oxygen species (ROS) by mitochondria. We tested whether preconditioning's physiological triggers, bradykinin and opioid, also signal through the EGFR. Both bradykinin and the synthetic delta-opioid agonist DADLE increased ROS production in isolated cardiomyocytes by approximately 50%. DADLE's effect was abrogated by either metalloproteinase inhibitor III (MPI) or the diphtheria toxin mutant CRM-197 which blocks heparin-binding EGF shedding indicating that DADLE signals through EGFR transactivation. MPI also blocked DADLE's infarct-sparing effect in whole hearts. Additionally, blocking Src kinase (a component of the EGFR's signaling complex) with PP2 or PI3-K with wortmannin blocked DADLE's effect on cardiomyocyte ROS production and PP2 blocked DADLE's salvage of ischemic myocardium. Finally, DADLE increased phosphorylation of Akt and extracellular signal-regulated protein kinases (ERK) 1/2 in left ventricular myocardium, and this increase was blocked by the EGFR antagonist AG1478. On the other hand, neither MPI nor CRM-197 prevented bradykinin from increasing ROS production, and MPI did not affect bradykinin's infarct-sparing effect in intact hearts. Conversely, both PP2 and wortmannin blocked bradykinin's effect on ROS generation and also aborted bradykinin's cardioprotective effect in intact hearts. While bradykinin also increased phosphorylation of Akt and ERK in myocardium, that increase was not affected by AG1478. Hence bradykinin, unlike acetylcholine or opioid, does not transactivate EGFR, although all 3 agonists do signal through Src and PI3-K.