To assess the role of the endothelial nitric oxide synthase (eNOS) gene variant as a risk factor for atherosclerosis we sought to investigate whether the Glu298Asp polymorphism of the eNOS gene is associated with functional changes in the endothelium in healthy volunteers. Methods—Endothelial function was assessed in 68 normal volunteers (ages 18–44 years) by bilateral forearm venous occlusion plethysmography with intraarterial infusions of increasing doses of acetylcholine for endothelial-dependent vasodilation and, with sodium nitroprusside and verapamil for endothelial-independent vasodilation. Blood was genotyped by polymerase chain reaction and BanII digestion. Results—Asp homozygotes (TT) had a decreased vasodilatory response to acetylcholine [forearm blood flow (FBF) ratio between infused and control arm, 2.82 ± 1.10] when compared to GG variant (FBF ratio to acetylcholine, 3.97 ± 1.90, p= 0.04) and to a certain extent, the GT variant (FBF ratio to acetylcholine, 3.79 ± 2.28, p= 0.07). There was no effect of eNOS genotype on the response to the endothelial-independent vasodilators—sodium nitroprusside and verapamil. Conclusions—Our data show that carriage of the Asp298 variant of the eNOS gene is associated with a blunted endothelial-dependent vasodilation in healthy volunteers. These findings support a genetically determined modulation of endothelial dysfunction, a phenotype of early atherosclerosis in humans.