The ubiquitous chemical messenger molecule nitric oxide (NO) has been implicated in a diverse range of biological activities including neurotransmission, smooth muscle motility and mediation of nociception. Endogenous synthesis of NO by the neuronal isoform of the nitric oxide synthase gene family has an essential role within the central and peripheral nervous systems in addition to the autonomic innervation of cerebral blood vessels. To investigate the potential role of NO and more specifically the neuronal nitric oxide synthase (nNOS) gene in migraine susceptibility, we investigated two microsatellite repeat variants residing within the 5' and 3' regions of the nNOS gene. Population genomic evaluation of the two nNOS repeat variants indicated significant linkage disequilibrium between the two loci. Z-DNA conformational sequence structures within the 5' region of the nNOS gene have the potential to enhance or repress gene promoter activity. We suggest that genetic analysis of this 5' repeat variant is the more functional variant expressing gene wide information that could affect endogenous NO synthesis and potentially result in diseased states. However, no association with migraine (with or without aura) was seen in our extensive case-control cohort (n = 579 affected with matched controls), when both the 5' and 3' genetic variants were investigated.