Background: Several neurotransmitters are expressed in the neurons of the trigeminal ganglion. One such signalling molecule is the pituitary adenylate cyclase-activating peptide (PACAP). PACAP signalling has been suggested to have a possible role in the pathophysiology of primary headaches. Objective: The present study was designed to investigate the relationship between PACAP and calcitonin gene-related peptide, currently the two most relevant migraine peptides. Methods: In the current study, we used ELISA to investigate PACAP and calcitonin gene-related peptide release in response to 60 mM K+ or capsaicin using a rat hemi-skull model. We combined this analysis with qPCR and immunohistochemistry to study the expression of PACAP and calcitonin gene-related peptide receptors and ligands. Results: Calcitonin gene-related peptide (CGRP) is released from the trigeminal ganglion and dura mater. In contrast, PACAP is only released from the trigeminal ganglion. We observed a weak correlation between the stimulated release of the two neuropeptides. PACAP-38 immunoreactivity was expressed alone and in a subpopulation of neurons in the trigeminal ganglion that also store calcitonin gene-related peptide. The receptor subtype PAC1 was mainly expressed in the satellite glial cells (SGCs), which envelop the neurons in the trigeminal ganglion, in some neuronal processes, inside the Ad-fibres and in the outermost layer of the myelin sheath that envelopes the Ad-fibres. Conclusion: Unlike CGRP, PACAP is only released within the trigeminal ganglion. This raises the question of whether a migraine therapy aimed at preventing peripheral PACAP signalling would be as successful as the CGRP signalling targeted treatments.