Long-term exposure to organic nitrates influences different sections of the vascular bed heterogeneously. Continuous dosage of nitrates leads to the development of tolerance both to the vascular effects and to the unwanted adverse effect, headache. Human data on the development of tolerance in different cranial arteries over more than 24 h are lacking. We compared the vascular changes of the middle cerebral, superficial temporal and radial arteries during oral administration of isosorbide-5-mononitrate (5-ISMN) 30 mg three times daily for 7 days in 11 healthy subjects in a double-blind, randomised, placebo controlled cross-over design. Blood velocity in the middle cerebral artery was measured with transcranial Doppler and the diameters of the temporal and radial arteries were measured with high frequency ultrasound. Headache recordings were compared to the observed vascular changes over time. Tolerance was complete within 24 h in the middle cerebral artery whilst in the superficial temporal and the radial arteries, tolerance was only partial and developed much more slowly, i.e. after 7 days correlating with the disappearance of NO-induced headache. The present study thus demonstrated the important differences in the time profiles of appearance of nitrate tolerance in arteries of different vascular beds in man. If vasodilatation is the cause of NO-induced headache the results point to extracerebral arteries as the locus of nociception. Due to a variety of other possible pain-inducing effects of nitric oxide our results do not exclude cerebral arteries.