The present study investigated the effect of tumor necrosis factor (TNF)-α on myocardial energy metabolism as estimated by myocardial oxygen consumption (MV˙o2). MV˙o2of electrically stimulated isolated trabeculae of right ventricular Wistar rat myocardium was analyzed using a Clark-type oxygen probe. After the initial data collection in the absence of TNF-α, measurements were repeated after TNF-α stimulation. In separate experiments, pretreatment with the nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) or the ceramidase inhibitor n-oleoylethanolamine (NOE) was done to investigate NO/sphingosine-related effects. TNF-α impaired myocardial economy at increasing stimulation frequencies without altering baseline MV˙o2. Incubation with TNF-α in the presence of l-NAME further impaired myocardial economy. NOE preincubation abrogated the TNF-α effect on myocardial economy. Moreover, the negative inotropic effect of TNF-α was observed in NOE-pretreated but not l-NAME-pretreated muscle fibers. Exogenous sphingosine mimicked the TNF-α effect on mechanics and energetics. We conclude that TNF-α impairs the economy of chemomechanical energy transduction primarily through a sphingosine-mediated pathway.