Nitric oxide (NO) generated from inducible NO synthase (iNOS) participates in immune and inflammatory responses in many tissues. The NO donor glyceryl trinitrate (GTN) provokes delayed migraine attacks when infused into migraineurs and also causes iNOS expression and delayed inflammation within rodent dura mater. Sodium nitroprusside, an NO donor as well, also increases iNOS expression. Because inflammation and iNOS are potential therapeutic targets, we examined transcriptional regulation of iNOS following GTN infusion and the consequences of its inhibition within dura mater. We show that intravenous GTN increases NO production within macrophages. L-N(6)-(1-iminoethyl)lysine, a selective iNOS inhibitor, attenuates the NO signal, emphasizing the importance of enzymatic activity to delayed NO production. iNOS expression is preceded by significant nuclear factor kappa B (NF-kappaB) activity, as reflected by a reduction in the inhibitory protein-kappa-Balpha (IkappaBalpha) and activation of NF-kappaB after GTN infusion. IkappaBalpha degradation, NF-kappaB activation, and iNOS expression were attenuated by parthenolide (3mg/kg), the active constituent of feverfew, an anti-inflammatory drug used for migraine treatment. These findings suggest that GTN promotes NF-kappaB activity and inflammation with a time course consistent with migraine attacks in susceptible individuals. We conclude, based on results with this animal model, that blockade of NF-kappaB activity provides a novel transcriptional target for the development of anti-migraine drugs.