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Background-The NADPH oxidase, by generating reactive oxygen species, is involved in the pathophysiology of many cardiovascular diseases and represents a therapeutic target for the development of novel drugs. A single-nucleotide polymorphism, C242T of the p22(phox) subunit of NADPH oxidase, has been reported to be negatively associated with coronary heart disease and may predict disease prevalence. However, the underlying mechanisms remain unknown. Methods and Results-With the use of computer molecular modeling, we discovered that C242T single-nucleotide polymorphism causes significant structural changes in the extracellular loop of p22(phox) and reduces its interaction stability with Nox2 subunit. Gene transfection of human pulmonary microvascular endothelial cells showed that C242T p22(phox) significantly reduced Nox2 expression but had no significant effect on basal endothelial O-2(center dot-) production or the expression of Nox1 and Nox4. When cells were stimulated with tumor necrosis factor-alpha (or high glucose), C242T p22(phox) significantly inhibited tumor necrosis factor-alpha-induced Nox2 maturation, O-2(center dot-) production, mitogen-activated protein kinases and nuclear factor kappa B activation, and inflammation (all P<0.05). These C242T effects were further confirmed using p22(phox) short-hairpin RNA-engineered HeLa cells and Nox2(-/-) coronary microvascular endothelial cells. Clinical significance was investigated by using saphenous vein segments from non-coronary heart disease subjects after phlebotomies. TT (C242T) allele was common (prevalence of approximate to 22%) and, in comparison with CC, veins bearing TT allele had significantly lower levels of Nox2 expression and O-2(center dot-) generation in response to high-glucose challenge. Conclusions-C242T single-nucleotide polymorphism causes p22(phox) structural changes that inhibit endothelial Nox2 activation and oxidative response to tumor necrosis factor-alpha or high-glucose stimulation. C242