Migraine is a primary headache disorder which involves both genetic and environmental components. Since angiotensin-converting enzyme (ACE) and matrix metalloproteinase (MMP) share the same homology, we investigated whether the MMP-3 and ACE I/D gene variants are involved in migraine risk and whether the ACE variant might act in combination with the MMP-3 genetic variant in patients with migraine. This is the first study to evaluate the association between MMP-3 and ACE polymorphisms, and migraine. Genotypes were determined by polymerase chain reaction. The frequencies of 5A5A genotypes of the MMP-3 and D allele of ACE were significantly elevated, but II genotypes of the ACE and 6A allele of MMP-3 significantly decreased in all patients. The combined DD/5A5A and ID/5A5A genotypes increased the risk of migraine. Individuals who were homozygous for the deletion (D) allele showed increased ACE activity. Subjects with the 5A5A genotype and/or D allele or with the combined DD/5A5A or ID/5A5A might be more susceptible to migraine development. In contrast, subjects with the II and/or 6A6A genotypes may be protected from migraine development. The greater activity of the 5A5A and DD genotypes might result in vascular reactivity that is more pronounced in migraine. Taken together, our data suggest that numerous genes may influence ACE activity. Discovery of new genes might better clarify the pathogenesis of migraine and open an avenue to therapeutic strategies against migraine.