The aim of this study was to investigate the possible association between the inducible nitric oxide synthase (NOS2) gene promoter polymorphism, CCTTTn microsatellite, with celiac disease susceptibility. We carried out a familial study in which 53 Spanish families were genotyped by a polymerase chain reaction (PCR)-based method combined with fluorescent technology. A transmission disequilibrium test was performed to investigate the transmission pattern of the different CCTTTn alleles from parents to affected offspring. The test did not reach any statistically significant difference because none of the CCTTTn repeats was shown to be significantly transmitted to the affected siblings. Our data suggest that the CCTTTn pentanucleotide microsatellite in the NOS2 gene promoter does not play a major role in celiac disease development.