Background: Cardiovascular events are the leading cause of morbidity and mortality in patients with end-stage renal disease. The role of endothelial dysfunction, an early marker of arteriosclerosis, in patients with chronic renal failure (CRF) before the initiation of maintenance hemodialysis (HD), and the factors affecting endothelial dysfunction in the setting of chronic renal failure remain poorly understood. Methods: We evaluated endothelial function by measuring flow-mediated vasodilation (%FMD) during reactive hyperemia in healthy individuals (HCS) and patients with chronic renal failure with (HD) or without (ND) hemodialysis. Nonspecific endothelium-independent vasodilation (%NTG) was measured after the administration of sublingual glyceryl trinitrate spray (0.3 mg). Factors affecting %FMD and %NTG were also tested. Results: In ND and HD, plasma homocysteine, cysteine and stable NO metabolite (NO<sup>–</sup><sub>3</sub>) concentrations were significantly elevated. In ND and HD, reactive hyperemia as well as %NTG and %FMD were attenuated to a similar degree. On multivariate regression analysis, NO<sup>–</sup><sub>3</sub> concentration was directly correlated with both %FMD and %NTG, while the glutathione (GSH) concentration correlated with only %NTG. Conclusion: Our findings indicate that chronic renal failure before the initiation of maintenance hemodialysis impairs endothelial function and/or the response to NO, which is accompanied by the attenuated reactive hyperemia. Furthermore, the impairment might be related to the decreased synthesis or the dissipation of NO.