Effects of TNF on nitric oxide (NO) production have been well documented in a variety of experimental and clinical settings, as for example, in septic shock. Investigations focusing on an inverse relation of NO on TNF synthesis are rare. Previously we could demonstrate that exogenous NO-releasing agents suppress LPS-induced TNF production in human PBMC. We now investigate whether a regulatory role on TNF synthesis could also be ascribed to endogenous NO. This was studied in the murine macrophage cell line RAW 264.7, which is able to express both TNF and the inducible NO synthase. No production was determined by measuring nitrite with Griess reagents. TNF formation was quantified by L929 cytotoxicity. We found a suppression of LPS-induced TNF synthesis by the exogenous addition of NO-releasing agents in the murine cell line, as previously observed in human cells. The application of NO synthase inhibitors led to a decrease in NO production, associated with an increase in TNF synthesis. TNF production increased from a base line (stimulation with 1 microgram/ml LPS alone) of 20.8 ng/ml to 36.3 ng/ml (means of six experiments) in the presence of the NO synthase inhibitor NG-monomethyl L-arginine (100 microM). Similar results were obtained with another NO synthase inhibitor, NG-nitro L-arginine-methylester. Lack of L-arginine in the medium resulted in a threefold increase in LPS-stimulated TNF synthesis compared with medium containing the usual concentration of 1 mM L-arginine. Restitution of L-arginine but not of D-arginine reversed this increase in TNF synthesis in a dose-dependent manner. To our knowledge these results indicate for the first time a negative feedback by endogenous NO on TNF synthesis in vitro. This finding may be relevant in pathophysiologic processes in which both TNF and NO are formed and in experimental therapies aiming at changes of NO concentrations.