Although hypercholesterolemia is known to impair endothelium-dependent vasodilation (EDV) long before the appearance of atherosclerotic plaques, it remains unclear whether the immune mechanisms that have been implicated in atherogenesis also contribute to the early oxidative stress and endothelial cell dysfunction elicited by HC. EDV (wire myography), superoxide generation (cytochrome C reduction), and NAD(P)H oxidase mRNA expression were monitored in aortic rings from wild type (WT) and mutant mice placed on either a normal or cholesterol-enriched diet (HC) for 2 weeks. WT mice on an HC diet exhibited an impaired EDV, enhanced superoxide generation, and increased expression of Nox-2 mRNA. The impaired EDV and increased superoxide generation induced by HC were significantly blunted in immunodeficient SCID mice and CD4+ T-lymphocyte deficient mice. These responses were also attenuated in HC mice that were genetically deficient in interferon- (IFN-), however adoptive transfer of WT-HC CD4+ T-lymphocytes to IFN--deficient recipients restored the HC-induced responses. The HC-induced impaired EDV and oxidative stress were also attenuated in HC mice that were genetically deficient in Nox-2 (gp91^phox-/-) and in WTgp91^phox-/--HC chimeras. HC-induced gp91^phox mRNA expression was significantly blunted in mice deficient in CD4+ T-cells or IFN-, and was restored with adoptive transfer of WT-HC CD4+ T-cells to IFN--deficient recipients. These findings implicate the immune system in the early endothelial cell dysfunction associated with hypercholesterolemia and are consistent with a mechanism of impaired EDV that is mediated by CD4+ T-cells and IFN-, acting through the generation of superoxide from vascular NAD(P)H oxidase.