Type 2 diabetes is associated with increased risk of atherosclerosis, yet the molecular mechanisms that underlie this remain poorly characterised. Endothelial dysfunction, characterised by reduced nitric oxide (NO) bioavailability, is a key early event in the development of atherosclerosis [1]. Insulin has been proposed to contribute to the maintenance of vascular health, and we and others have demonstrated that insulin stimulates NO synthesis in cultured human endothelial cells via PKB/Akt-mediated phosphorylation of endothelial NO synthase (eNOS) at Ser615 and Ser1177 [2,3]. Increased levels of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNFα) are observed in type 2 diabetes, and TNFα has previously been reported to inhibit insulin-stimulated NO synthesis and eNOS Ser1179 (equivalent to Ser1177) phosphorylation in bovine endothelial cells [2], yet the regulation of insulin-stimulated NO synthesis by TNFα in human endothelial cells remains poorly characterised. We therefore investigated the regulation of insulin-stimulated signalling pathways and NO synthesis by TNFα in cultured human aortic endothelial cells (HAECs). HAECs were preincubated with TNFα (10 ng/ml) for 0.5 or 6h prior to stimulation with insulin (15min, 1 μmol/l). NO synthesis and phosphorylation of insulin signalling proteins was assessed as described previously using a Sievers 280A NO meter and by immunoblotting respectively [3]. Preincubation of HAECs with TNFα for either 30min or 6h significantly inhibited insulin-stimulated NO production (40.4±4.9% and 30.8±15.9% respectively, n=3, p<0.05), yet only preincubation for 6h with TNFα was associated with reduced insulin-stimulated phosphorylation of Akt at Ser473 and eNOS at Ser1177 (55.0±9.3% and 34.3±12.6% respectively, n=4, p<0.05). As these data indicate reduced insulin-stimulated NO synthesis in the absence or reduced insulin-stimulated eNOS phosphorylation after 0.5h preincubation with TNFα, we examined the role of reactive oxygen species (ROS) in the acute action of TNFα. Incubation of HAECs with TNFα for either 0.5h or 6h significantly stimulated NADPH-dependent lucigenin chemiluminescence (414±139% and 230±63% respectively, n=3, p<0.05), and 30 min preincubation with the antioxidant tiron (1mmol/l) or gp91 ds-tat (50 μmol/l), an inhibitor of NADPH oxidase, ablated the inhibition of insulin-stimulated NO synthesis by TNFα. Taken together, these data indicate that TNFα inhibits insulin-stimulated NO synthesis via a mechanism involving the NADPH oxidase-mediated stimulation of ROS and that only prolonged TNFα stimulation causes any significant downregulation of the insulin-stimulated eNOS phosphorylation via Akt. Inhibition of insulin-stimulated NO synthesis by TNFα by these mechanisms may contribute to endothelial dysfunction in subjects with type 2 diabetes.