The aim of the study was to compare the effects of three eNOS gene polymorphisms associated with congenital heart defects, between Down syndrome patients with and without cardiac anomalies. Transthoracic echocardiographic examinations and eNOS single-nucleotide polymorphisms were investigated on seventy-five patients, prospectively. The frequencies of mutant alleles in the eNOS promoter (the -786T/C polymorphism) and exon 7 mutant alleles (the 894G--->T polymorphism) were significantly higher in Down syndrome patients with and without cardiac anomalies. The frequency of the intron GIOT polymorphism did not significantly differ between patients with and without cardiac anomalies. We found a significant relationship between eNOS gene polymorphisms and the congenital heart defects in patients with Down syndrome. Screening for the presence or absence of eNOS polymorphisms may be useful to obtain preliminary data on the risk of congenital heart defects in patients with Down syndrome.