OBJECTIVES: The present study aimed to investigate the influence of endogenous tumor necrosis factor-alpha (TNF-α) that was synthesized during ischemia and exogenous TNF-α on endothelial and inducible nitric oxide synthase (eNOS and iNOS) messenger ribonucleic acid (mRNA) expression and nitric oxide (NO) production in the isolated rat heart. BACKGROUND: Tumor necrosis factor-α is recognized as being a proinflammatory cytokine with a significant cardiodepressant effect. One of the proposed mechanisms for TNF-α-induced cardiac contractile dysfunction is increased NO production via iNOS mRNA upregulation, but the role of NO in TNF-α-induced myocardial dysfunction is highly controversial. METHODS: Isolated rat hearts studied by a modified Langendorff model were randomly divided into subgroups to investigate the effect of 1-h global cardioplegic ischemia or the effect of 1-h perfusion with exogenous TNF-α on the expression of eNOS mRNA and iNOS mRNA and on NO production. RESULTS: After 1 h of ischemia, there were significant increases in TNF levels in the effluent (from hearts), and eNOS mRNA expression had declined (from 0.91 ± 0.0g to 0.68 ± 0.19, p < 0.001); but there were no changes in iNOS mRNA expression, and NO was below detectable levels. Perfusion of isolated hearts with TNF-a had a cardiodepressant effect and decreased eNOS mRNA expression to 0.67 ± 0. 04 (p < 0.002). Inducible nitric oxide synthase mRNA was unchanged, and NO was below detectable levels. CONCLUSIONS: We believe this is the first study to directly show that TNF-α does not increase NO synthesis and release but does downregulate eNOS mRNA in the ischemic and nonischemic isolated rat heart.