Recent studies have shown that cytokines and endotoxins impair insulin-stimulated glucose transport by activating the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in skeletal muscle cells. In this study, we investigated whether iNOS induction is modulated by insulin in L6 myocytes. Long term exposure of muscle cells to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) greatly increased iNOS mRNA expression and NO production. Addition of insulin to the cytokine/LPS-treated muscle cells reduced (by approximately 40%) NO production. This inhibition was similar to that observed with the synthetic glucocorticoid dexamethasone, a known inhibitor of iNOS in several cell types. The combination of insulin and dexamethasone was more effective than either agent alone in reducing NO production. Dexamethasone greatly inhibited the effect of cytokines/LPS to induce cellular iNOS mRNA expression. In strong contrast, insulin failed to reduce iNOS mRNA expression under similar conditions. These results show that insulin is a novel inhibitor of iNOS-mediated NO production in skeletal muscle cells. Furthermore, our data indicate that unlike glucocorticoids, insulin does not inhibit NO production by suppression of iNOS gene transcription.