Stimulation of cardiac ₂-adrenergic receptor (₂-AR) or -opioid receptor (DOR) exerts a similar degree of cardioprotection against myocardial ischemia in experimental models. We hypothesized that -opioid-initiated cardioprotection is mediated by the intrinsic cardiac adrenergic (ICA) cell via enhanced epinephrine release. Using immunohistochemical and in situ hybridization methods, we detected in situ tyrosine hydroxylase (TH) mRNA and TH immunoreactivity which was co-localized with DOR immunoreactivity in ICA cells in human and rat hearts. Western blot analysis detected DOR protein in ICA cells isolated from rat ventricular myocytes. The physiology of DOR expression was examined by determining changes of cytosolic [Ca²⁺]i transients in isolated rat ICA cells using fluorescence spectrophotometry. Exposing the selective -opioid agonist DPDPE to ICA cells increased [Ca²⁺]i transients in a concentration-dependent manner. Such an effect was abolished by the Ca²⁺ channel blocker nifedipine. HPLC-electrochemical detection demonstrated a 2.4-fold increase in epinephrine release from ICA cells following DPDPE application. The significance of ICA cell and its epinephrine release in -opioid-initiated cardioprotection was demonstrated in the rat myocardial infarction model and ICA cell-myocyte co-culture. DPDPE administered before coronary artery occlusion or simulated ischemia-reperfusion reduced left ventricular infarct size by 54±15% or myocyte death by 26±4%, respectively. ₂-AR blockade markedly attenuated -opioid-initiated infarct-size-limiting effect and abolished -opioid-initiated myocyte survival protection in rat ICA cell-myocyte co-culture. Furthermore, -opioid agonist exerted no myocyte survival protection in the absence of co-cultured ICA cells during ischemia-reperfusion. We conclude that -opioid-initiated cardioprotection is primarily mediated via endogenous epinephrine-₂-AR signaling pathway as a result of ICA cell activation.