Association of the three potential endothelial nitric oxide synthase gene (eNOS) polymorphisms (T-786C in promoter region, G894T in exon 7 and tandem 27-bp repeats in intron 4) with an increased risk of lacunar infarction (LI) were investigated. Genotypes of 70 patients and 81 healthy controls were determined through PCR with or without RFLP. Flow-mediated dilatation (FMD) was performed to assess endothelial-dependent vasodilatation, whereas the endothelial-independent vasodilatation was assessed with nitroglycerin (NTG). Genotype distribution was significantly different between LI patients and controls for intron 4aa (alleles for four repeats), genotype frequency being 1.4% and 16.0%, respectively (odds ratio for additive effect, 0.47; 95% CI, 0.28-0.81; p=0.006). Haplotypes with the intron 4aa polymorphism were significantly higher in controls when compared with the LI group (p=0.001). Diminished FMD but normal NTG response confirmed that patients with LI have generalized endothelial dysfunction. Intron 4aa genotype of eNOS gene seems to be protective for isolated LI and the effect was potentiated by the absence of 786C polymorphism in any allele of the promoter region.