Nitrovasodilators that produce nitric oxide (NO) in the organism are known to cause delayed headaches in migraineurs accompanied by an increase in plasma levels of calcitonin gene-related peptide (CGRP) and NO metabolites in the venous outflow of the head. The same treatment induces increased activity of neurons in the spinal trigeminal nucleus in a rat model of meningeal nociception. To examine if these changes induced by NO are associated with an increase in CGRP and NO producing trigeminal afferents, immunohistochemistry was utilised. The NO donor glyceryl trinitrate (GTN, 250 mg/kg) or vehicle was i.v. infused for 2 hours into isofluorane anaesthetised rats. After additional 4 hours under anaesthesia the animals were fixed by paraformaldehyde perfusion, the trigeminal ganglia were dissected and series of cryosections were immunostained for detection of CGRP and neuronal NO synthase (nNOS). The trigeminal ganglion neurons showing immunofluorescence for either these proteins were counted. The proportions of both CGRP- and nNOS-immunopositive neurons as well as neurons showing both these markers were significantly increased after SNP compared to vehicle treatment in all three divisions of the trigeminal ganglia. We conclude that high levels of NO induce the expression of CGRP and NO-producing enzymes in a feed-forward manner. Similar changes may be involved in the nitrovasodilator-induced as well as spontaneous headache attacks in migraineurs.