5-HT1D (but not 5-HT1B)-receptor immunoreactivity (i.r.) can be detected on trigeminal fibres within the spinal trigeminal tract of the human brainstem. The present study used immunohistochemical and morphometric techniques to determine the proportions of trigeminal fibres expressing substance P, CGRP or 5-HT1D-receptor immunoreactivities. Co-localization studies between 5-HT1D-receptor and substance P- or CGRP-i.r. were also performed. Brainstem material was obtained with consent (four donors) and the total number of immunoreactive fibres within the trigeminal tract was estimated using random field sampling. A greater proportion of fibres (>1 microm diameter) expressed CGRP-i.r. (80 +/- 6%) compared with substance P-i.r. (46 +/- 7%) or 5-HT1D-receptor-i.r. (25 +/- 1%). 5-HT1D-receptor-i.r. was co-localized on some CGRP- or substance P-i.r. fibres. This suggests that 5-HT1D-receptors can regulate the release of CGRP and substance P and may be relevant to the clinical effectiveness of 5-HT1B/1D-receptor agonists in the treatment of migraine and other cranial pain syndromes.