Several biochemical, neuropathological, and experimental data suggest a possible role of nitric oxide (NO) in the pathophysiology of restless legs syndrome (RLS). Two single nucleotide polymorphisms (SNPs) neuronal nitric oxide synthase (nNOS or NOS1) gene (rs7977109 and rs693534) have been found to be associated with the risk for RLS in Germans, although only one of them (rs7977109) remained as significant after multiple comparison tests. The aim of our study was to replicate the possible association between these SNPs and risk for RLS in the Spanish population. We studied the allelic and genotype frequencies of the SNPs rs7977109 and rs693534 in 205 patients with RLS and 328 healthy controls using TaqMan genotyping. The rs7977109 and rs693534 genotypes and allelic frequencies did not significantly differ between patients with RLS and controls and were unrelated with the age at onset of RLS, gender, ferritin levels, and response to dopaminergic or gabaergic agents. The rs7999109GA genotype was overrepresented in RLS patients with positive family history of RLS, and in patients with symptomatic response to clonazepam. The results of our study suggest that these two NOS1 SNPs are not related to the overall risk for RLS in the Spanish population.