Background:  Several findings suggest that nitric oxide (NO) plays a significant role in the regulation of the Th1/Th2 balance and contributes to the development of allergic diseases. Our study investigates a possible association of C/T transition located 276‐bp downstream from the translation termination site in exon 29 of the human nitric oxide synthase type 1 (NOS1) gene with immunoglobulin E (IgE)‐mediated allergic diseases in the Czech population.Methods:  The study included 688 subjects – 368 patients with clinically manifested allergic diseases and 320 unrelated controls with negative familial history of asthma/atopy. The NOS1 genotypes were determined by polymerase chain reaction (PCR) and restriction analysis by Eco72I.Results:  No significant differences were found for allele or genotype frequencies of the 5266 C/T polymorphism in exon 29 of the NOS1 gene between IgE‐mediated allergic diseases (or asthma alone) and healthy subjects. However, this common polymorphism showed a significant association with signs of atopy, especially with total serum IgE levels [loge IgE levels (mean ± SD): CC genotype = 4.34 ± 1.40; CT genotype = 4.58 ± 1.53; TT genotype = 5.01 ± 1.61; P < 0.05).Conclusions:  Our findings suggest that NOS1 gene may participate in the pathogenesis of high total serum IgE levels in allergic diseases in our population. These findings provide support for NOS1 as a candidate gene for IgE‐mediated allergy.