An endothelial nitric oxide synthase (eNOS) gene polymorphism (Glu298Asp) has been associated with cardiovascular disease. We investigated whether carriage of the polymorphism was associated with functional changes in the endothelium, and how genotype altered the harmful and beneficial impact of environmental influences on the endothelium. Endothelium-dependent, flow-mediated brachial artery dilatation (FMD) and endothelium-independent dilatation response to glyceryl trinitrate were measured using high-resolution ultrasound in 248 subjects (131 female, 117 male, aged 20 to 28) genotyped for the Glu298Asp polymorphism. Vascular function was compared between genotype groups and interactions with the proatherogenic risk factor, smoking, and the antiatherogenic influence of n-3 fatty acids (n-3FA) were investigated. Vascular function was not related to genotype in the group as a whole or within sexes. However, among males, smoking was associated with lower FMD in Asp298 carriers (nonsmokers 0.125±0.085 mm versus smokers 0.070±0.060 mm,P=0.006) but not in Glu298 homozygotes (nonsmokers 0.103±0.090 mm versus smokers 0.124±0.106,P=0.5). In the whole group, n-3FA levels were positively related to FMD in Asp298 carriers (reg coeff=0.023 mm/%,P=0.04,r=0.20) but not in Glu298 homozygotes (reg coeff=−0.019 mm/%,P=0.1). These differences between genotype groups were significant in interaction models. The Glu298Asp polymorphism is associated with differences in endothelial responses to both smoking and n-3 FA in healthy young subjects. These findings raise the possibility of genotype-specific prevention strategies in cardiovascular disease.