The association between endothelial constitutive nitric oxide synthase (ecNOS) gene polymorphism and vascular endothelial function has not been clarified. We investigated the impact of ecNOS gene polymorphism on endothelial function in 95 patients with Type 2 diabetes (ecNOS genotype: 4b/b, n = 62; 4b/a, n = 30; 4a/a, n = 3). Flow-mediated (endothelium dependent, FMD) and nitroglycerin-induced (endothelium independent, NTG) vasodilations of the right brachial artery were studied using a phase-locked echotracking system. There were no significant differences in clinical characteristics among the ecNOS genotypes. The FMD was significantly lower in the patients with ecNOS4a allele than in those without ecNOS4a allele (P < 0.05). Multiple regression analysis showed that ecNOS4a allele and mean blood pressure were significant independent determinants for reduced FMD in all patients (R(2) = 0.122, P = 0.0025). The ecNOS4a allele was an independent determinant for reduced FMD in smokers but not in nonsmokers. These results suggest that ecNOS4a allele is a genetic risk factor for endothelial dysfunction in diabetic patients, especially in smokers.