Nitric oxide (NO) derived from endothelial cells is profoundly related to the maintenance of physiological vascular tone. Impairment of endothelial NO generation brought about by gene polymorphism is considered one of the deterioration factors for progressive renal disease. In the eNOS intron 4 polymorphism there are two alleles a and b are: a has four and b 5 five tandem 27-bp repeats. The presence of aa genotype has been associated with cardiovascular and renal disease. The aim of this study was to investigate the potential involvement of endothelial NO synthase (eNOS) gene intron 4 a/b variable number of tandem repeats (VNTR) polymorphism in patients with end-stage renal disease (ESRD). A total of 131 individuals, 37 diabetic nephropathy ESRD patients and 94 controls were involved in this case-control study. Genomic DNA was isolated from peripheral blood leukocytes using DNA extraction kit (QIAamp DNA Blood Mini Kit (250) – Qiagen- Valencia, California, USA). For polymerase chain reaction (PCR) amplification, two oligonucleotide primers were used that flank the region of the 27 bp repeat sequence in intron 4 of the ecNOS gene. The eNOS primers were synthesized by Integrated DNA Technologies, Inc (IDT). The forward primer was 5'-AGGCCCTATGGTAGTGCCTTT-3' and the reverse primer was 5'-TCTCTTAGTGCTGTGGTCAC-3'. Genomic DNA (100 ng) was amplified by PCR using 10 m L of Master Mix (Eppendorf, Hamburg-Germany), 10 pmoL of each primer in a final reaction volume of 25 µL. The reaction mixture was heated to 94°C for 6 min for denaturation and then subjected to 35 cycles at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 2 min. Final extension was at 72°C for 7 min. The PCR products were analysed by electrophoresis in 2.5% agarose gels stained with ethidium bromide.No significant deviation from the Hardy-Weinberg equilibrium was observed in control and case population (c ²=0.186, and c ²=0.02). The frequencies of eNOS genotypes aa, ab, bb in diabetic nephropathy patients and control group were: 9.4, 41.9 and 48.8% and 5.3, 30.9 and 63.8% respectively. It was found a significant difference between eNOS aa genotype frequency in patients and controls (P=0,012). The frequency of eNOSa allele as well as that of carries genotypes (eNOS4aa and eNOS4ab) was higher in diabetics patients than in healthy controls (P=0.0455, P=0.0152). The multiple logistic regression analysis revealed that carriers genotype were significantly related with a development of chronic renal failure (P=0.034; OR: 2.28 95% IC 1.04 – 5.00).This study shows a strong correlation between eNOS4a polymorphism and diabetic nephropathy.