Aging is associated with impaired vascular endothelial function, as indicated in part by reduced endothelium-dependent dilation (EDD). Decreased EDD with aging is thought to be related to vascular endothelial cell oxidative stress, but direct evidence is lacking. We studied 95 healthy men: 51 young (23+/-1 years) and 44 older (63+/-1 years). EDD (brachial artery flow-mediated dilation) was approximately 50% lower in older versus young men (3.9+/-0.3% versus 7.6+/-0.3%, P<0.01; n=42 older/n=51 young). Abundance of nitrotyrosine (quantitative immunofluorescence), an oxidatively modified amino acid and marker of oxidative stress, was higher in endothelial cells (ECs) obtained from the brachial artery (1.25+/-0.12 versus 0.61+/-0.11 nitrotyrosine intensity/human umbilical vein EC [HUVEC] intensity, P=0.01; n=11 older/n=11 young) and antecubital veins (0.55+/-0.04 versus 0.34+/-0.03, P<0.05; n=19 older/n=17 young) of older men. Flow-mediated dilation was inversely related to arterial EC nitrotyrosine expression (r=-0.62, P=0.01; n=22). In venous samples, EC expression of the oxidant enzyme NAD(P)H oxidase-p47(phox) was higher in older men (0.71+/-0.05 versus 0.57+/-0.05 NAD[P]H oxidase-p47(phox) intensity/HUVEC intensity, P<0.05; n=19 older/n=18 young), whereas xanthine oxidase and the antioxidant enzymes cytosolic and mitochondrial superoxide dismutase and catalase were not different between groups. Nuclear factor-kappaB p65, a component of the redox-sensitive nuclear transcription factor nuclear factor-kappaB, was elevated in both arterial (0.73+/-0.07 versus 0.53+/-0.05 NF-kappaB p65 intensity/HUVEC intensity, P<0.05; n=9 older/n=12 young) and venous (0.65+/-0.07 versus 0.34+/-0.05, P<0.01; n=13 older/n=15 young) EC samples of older men and correlated with nitrotyrosine expression (r=0.51, P<0.05 n=16). These results provide direct support for the hypothesis that endothelial oxidative stress develops with aging in healthy men and is related to reductions in EDD. Increased expression of NAD(P)H oxidase and nuclear factor-kappaB may contribute to endothelial oxidative stress with aging in humans.