Over-production of tumor necrosis factor-alpha (TNF-α) following myocardial ischemia-reperfusion contributes to cardiac dysfunction, and anti-TNF-α has therapeutic potential for myocardial protection in cardiac surgery with obligatory ischemia. It remains unclear, however, whether myocardial TNF-α production occurs during ischemia and whether cardiac myocytes constitute a source of myocardial TNF-α. Ischemia alone has been shown to activate myocardial NF-κB. We hypothesized that ischemia alone is sufficient to induce myocardial TNF-α gene expression and peptide synthesis. We examined TNF-α production and NF-κB activation in the isolated rat heart subjected to global normothermic ischemia. Myocardial ischemia resulted in rapid IκB-α degradation and NF-κB activation. Immunofluorescence staining detected NF-κB intranuclear translocation primarily in myocardial interstitial cells. Ischemia alone induced a time-dependent increase in myocardial TNF-α. TNF-α peptide increased to 20.3±3.0 pg/mg after 25 min of ischemia (P< 0.05 vs 8.9±2.0 pg/mg in perfusion control). TNF-α was also localized to myocardial interstitial cells. Increased TNF-α peptide level correlated with TNF-α mRNA expression. We conclude that ischemia alone induces TNF-α gene expression and peptide synthesis in the myocardium that are associated with NF-κB activation. Non-myocytes constitute the main source of myocardial TNF-α following ischemia. The results suggest that therapeutic strategies attempting to decrease myocardial TNF-α production need to be applied before or in the early phase of ischemia.