Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) mediates endothelium-dependent vasodilatation and antithrombotic action. Controversial results regarding the association of eNOS gene (NOS3) polymorphisms with hypertension have been reported. In the present study, we examined a possible association between the 27-base pair (bp) repeat polymorphism in intron 4 of the NOS3 gene and hypertension in a sample of the Tunisian population.A total of 295 Tunisian patients with hypertension and 395 healthy controls were included in the study. The NOS3 gene intron 4a4b variable number of tandem repeats polymorphism was analyzed by PCR.A significant differences in genotype distribution and allele frequency was observed between patients and controls. Patients with hypertension had a frequency of 6.4% for the 4a4a genotype, 32.7% for the 4a4b genotype and 60.9% for the 4b4b genotype. The controls had a frequency of only 2.3% for the 4a4a genotype, 28.4% for the 4a4b genotype and 69.4% for the 4b4b genotype (chi(2)=11.81, p=0.003). The hypertension patient group showed a significant higher frequency of the 4a allele compared to the controls (0.23 vs. 0.16; chi(2)=8.61, p=0.003). The odds ratio of hypertension for 4a vs 4b allele frequencies was statistically significant 1.66 [1.09-2.53] at 95% CI, p=0.01 in males, whereas it was non-significant in females (1.23 [0.84-1.81], p=0.26).The present study showed a significant and independent association between the NOS34a4b gene polymorphism (presence of 4a allele) and hypertension in the Tunisian population.