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Nitric oxide (NO) is known to be critically involved in breast carcinogenesis. Genetic polymorphisms of the gene encoding for endothelial nitric oxide synthase (Nos3), the enzyme catalyzing the production of the NO, are known to predispose to malignant disease. Whether these polymorphisms also influence breast cancer risk is unknown. In the present case-control study, we ascertained 2 polymorphisms of the Nos3 gene cluster (Nos3 exon 7 Glu298 Asp and a 27-base pair repeat in intron 4 of Nos3) in 269 Caucasian patients with breast cancer and 244 healthy controls using pyrosequencing and PCR, respectively. Presence of the exon 7 Nos3 polymorphism predisposed women to breast cancer (p = 0.03, Odds ratio [95% Confidence Intervals] = 1.9 [1.1-3.6]), but was not associated with any clinico-pathological parameters. No significant associations were ascertained with respect to the intron 4 Nos3 polymorphism. In our series, presence of the mutant exon7 Nos3 polymorphism was associated with an increased risk for breast cancer in Caucasian women.