To define the mechanism(s) responsible for the negative inotropic effects of tumor necrosis factor-alpha (TNFalpha) in the adult heart, we examined the functional effects of TNFalpha in the intact left ventricle and the isolated adult cardiac myocyte. Studies in both the ventricle and the isolated adult cardiac myocyte showed that TNFalpha exerted a concentration- and time-dependent negative inotropic effect that was fully reversible upon removal of this cytokine. Further, treatment with a neutralizing anti-TNFalpha antibody prevented the negative inotropic effects of TNFalpha in isolated myocytes. A cellular basis for the above findings was provided by studies which showed that treatment with TNFalpha resulted in decreased levels of peak intracellular calcium during the systolic contraction sequence; moreover, these findings did not appear to be secondary to alterations in the electrophysiological properties of the cardiac myocyte. Further studies showed that increased levels of nitric oxide, de novo protein synthesis, and metabolites of the arachidonic acid pathway were unlikely to be responsible for the TNFalpha-induced abnormalities in contractile function. Thus, these studies constitute the initial demonstration that the negative inotropic effects of TNFalpha are the direct result of alterations in intracellular calcium homeostasis in the adult cardiac myocyte.