Hypertension and high serum cholesterol level are important risk factors for atherosclerosis and coronary heart disease. In the present study we tested the hypothesis whether high sodium intake, when given in combination with Western type high-fat diet, induces endothelial dysfunction and promotes atherosclerosis. Furthermore, the role and enzyme sources of increased oxidative stress were examined. Low-density lipoprotein receptor-deficient mice (LDLR(-/-)) and control C57Bl/6 mice received either high-fat, normal-sodium diet (fat 18% and cholesterol 0.5%; NaCl 0.7%; w/w) or high-fat, high-sodium diet (7% NaCl w/w) for 12 weeks. Superoxide formation was assessed by lucigenin enhanced chemiluminescence, endothelial functions were examined ex vivo, and atherosclerotic lesions from the aorta were assessed by light microscopy. High-fat, high-sodium diet increased systolic blood pressure in LDLR(-/-) mice but not in C57Bl/6 mice, whereas it induced cardiac hypertrophy in both mouse strains. Dietary combination of fat and sodium induced endothelial dysfunction in LDLR(-/-) mice. Preincubation with a superoxide scavenger Tiron normalized endothelial dysfunction, whereas the hydrogen peroxide scavenger catalase did not alter endothelial function. High sodium intake induced superoxide formation in LDLR(-/-) mice on high-fat diet. Stimulation of muscarinic receptors in the endothelial cells by acetylcholine increased superoxide generation, whereas preincubation with the nitric oxide synthase (NOS) inhibitor L-arginine methyl ester or endothelium removal reduced superoxide production. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by apocynin decreased vascular superoxide formation whereas the xanthine oxidase inhibitor oxypurinol did not significantly affect oxidative stress in LDLR(-/-) mice. In conclusion, the detrimental effects of dietary sodium on endothelial function and progression of atherosclerosis in LDLR(-/-) mice on high-fat diet are mediated by increased ROS formation mainly through uncoupled NOS and NADPH oxidase. The present study also underscores the importance of superoxide and endothelial NOS uncoupling in the pathogenesis of endothelial dysfunction.