Recent studies suggest that nitric oxide (NO) plays an important role in nitrate-induced headache and in spontaneous migraine attacks. Organic nitrates act as prodrugs for NO and headache is a predominant adverse effect of nitrates but often disappears during continuous treatment. Insight into tolerance to headache could lead to insight into vascular headache mechanisms in general. The specific aim of the present study was therefore to characterize the headache and accompanying symptoms during continuous nitrate administration until a state of tolerance to headache had developed. 5-isosorbide-mononitrate (5-ISMN) 30 mg three times daily was administered orally for 7 days in 11 healthy subjects in a double-blind, randomized placebo controlled cross-over design. Wash-out between periods was 14 days or more. Haemodynamic data from the present study were compared to the observed changes of headache over time. Headache during 5-ISMN was longer lasting and more severe compared to placebo (P<0.004). In 10 subjects the headache fulfilled the pain sub-criteria for migraine and in five subjects all diagnostic criteria for migraine without aura were fulfilled. Conversely, 20 min of intravenous infusion of glyceryl trinitrate caused a milder headache and no migraine. The present results therefore suggest that NO may elicit a migraine attack in many healthy subjects if a high enough dose is given for several hours. A close temporal association between the disappearance of headache and the attenuation of the 5-ISMN induced dilatation of the superficial temporal artery was observed. In contrast, tolerance in the middle cerebral artery already appeared after 24 h, which was earlier than the development of tolerance to headache. If vasodilatation is the cause of headache the results point to extracerebral arteries. However, cytotoxic and pain modulating central nervous system effects of NO, the time courses of which are unknown, may also play a role, involving both intra- and extracranial arteries.