We investigated the association of 14 polymorphisms in the endothelial nitric oxide synthase gene (NOS3) with ankle brachial index (ABI) in non-Hispanic white hypertensives belonging to hypertensive sibships. Subjects (n=659, mean age 61+/-9 years, 54% women) underwent measurement of ABI using a standard protocol, and the lowest of 4 ABI values was used in the analyses. Non-synonymous SNPs with a minor allele frequency >0.02 and tag SNPs selected based on a measure of linkage disequilibrium (r(2)) were genotyped. We reduced the chance of false positives by testing for replication, randomly selecting 1 hypertensive sib from each sibship to create Subset 1 (n=330) and Subset 2 (n=329). Multivariable linear regression models were used to assess the associations of single NOS3 polymorphisms and haplotypes with ABI after adjustment for covariates (age, sex, body mass index, smoking, total cholesterol, HDL cholesterol, and diabetes). Two specific SNPs in significant LD with each other (rs891512 and rs1808593) were significantly associated with ABI in both subsets. Based on a sliding window approach with a window size of 2, estimated haplotypes from 2 SNP pairs (rs2070744-rs3918226 and rs1808593-rs7830) were also significantly associated with ABI in both subsets. In conclusion, specific NOS3 SNPs and haplotypes were associated with inter-individual variation in ABI, a non-invasive marker of peripheral arterial disease, in replicate subsets of hypertensive subjects. These findings motivate further investigation of the role of NOS3 variants in determining susceptibility to peripheral arterial disease.