Abstract. Treatment of neonatal rat cardiomyocytes for 72h in the presence of tumor necrosis factor (TNF) (10U/ml) lead to a decrease in basal and 1-adrenoceptor-induced formation of the calcium-mobilizing second messenger inositol trisphosphate (IP3) and its metabolites, IP2 and IP1, by 35 and 26%, respectively. The synthesis of phosphatidylinositol bisphosphate (PIP2), the substrate of PI-specific phospholipase C, was decreased by 45% following the TNF (10U/ml) exposure. Time courses of TNF (10U/ml)-induced alterations in rat cardiomyocytes showed a parallel decline of basal inositol phosphate formation and PIP2 synthesis suggesting that the decrease in inositol phosphate formation was due to the reduction in PIP2 synthesis. As the TNF-induced decrease of PIP2 synthesis was associated with a decreased synthesis of the phospholipid phosphatidylinositol (PI), the precursor of PIP2, by 33%, the decreased availability of PIP2 is apparently, at least in part, the result of the decreased synthesis of PI. As an apparent functional consequence of the decrease in IP3 formation following the TNF exposure, the 1-adrenoceptor-mediated induction of arrhythmias by 100mol/l noradrenaline+10mol/l timolol was abolished in TNF-pretreated rat cardiomyocytes. To investigate one of the possible mechanisms of the TNF-induced decrease of PIP2 formation, the effect of TNF pretreatment on glycerol-3-phosphate dehydrogenase (GDH), a key enzyme of lipogenesis, was studied: Exposure of the rat cardiomyocytes for 72h to TNF induced a concentration-dependent decrease in GDH activity by maximally 55%. The result presented are consistent with the hypothesis that the decreased basal and 1-adrenoceptor-induced formation of the second messenger IP3 observed in chronic endotoxinemia and sepsis may be mediated by a TNF-induced decrease in the synthesis of PIP2, the substrate of PI-specific phospholipase C. This mechanism occurs following long-term exposure to low TNF concentrations and is apparently distinct from the short-term cardiac effects induced by high concentrations of TNF.