Angiotensin II and nitric oxide play an important role in the function of arterial system. We wondered whether the mutations of angiotensin II type 1 receptor (AGTR1) and endothelial nitric oxide synthase (eNOS) genes are associated with increased stiffness of large arteries. Two frequent polymorphisms, A¹¹⁶⁶C of AGTR1 and T⁷⁸⁶C of eNOS, were assessed in a random, population-based sample of 250 subjects aged 25 to 64 years. Pulse wave velocity was measured in the aorta (APWV, between carotid and femoral arteries) and on the lower extremity (peripheral pulse wave velocity, PPWV, between femoral and tibialis posterior/dorsalis pedis arteries). Both polymorphisms were significantly associated with PPWV: 12.4±0.7, 13.8±0.2, 15.2±2.7 m/s for AA, AC and CC genotypes of AGTR1, respectively, p < 0.02 for trend; 12.3±0.8, 13.4±1.0, 15.1 ±1.6 m/s for TT, TC and CC genotypes of eNOS, respectively, p < 0.05). The combined effect of the polymorphisms was further studied. Subjects with 3–4 mutant alleles (heterozygous + homozygous or homozygous + homozygous, n = 35) had signigicantly increased PPWV (17.9±2.4 m/s) than those with no mutant allele (12.4±1.2 m/s) or 1–2 alleles (12.3±0.5 m/s, p < 0.007 for difference). These associations remained highly significant in multiple regression models with adjustment on potential confounders. The polymorphisms did not influence APWV or blood pressure. In conclusion, both AGTR1 and eNOS gene polymorphisms are associated with increased stiffness of peripheral muscular-type large arteries and their effect is synergistic. This finding reflects an interaction between the renin-angiotensin and nitric oxide systems in their effect on arterial properties.