Muscarinic acetylcholine receptors (mAChRs) have been widely reported as pharmacological targets for the treatment of pain. However, most of these efforts have focused on CNS mAChRs and their role in modulating nociception at the level of the spinal cord. The present study examines the contribution of peripheral mAChRs in trigeminal nociceptive pathways using a combination of in vivo and in vitro approaches. In the formalin model of orofacial nociception in rats, a peri-oral co-injection of the M2 agonist arecaidine dose-dependently inhibited phase 2 nocifensive behavior up to approximately 50% at 5 nmol. This effect was blocked by co-treatment with the mAChR antagonist atropine and was not seen when arecaidine was administered under the skin of the back, a site distant from that of the formalin injection. In vitro superfusion of isolated rat buccal mucosa with the non-selective mAChR agonist muscarine or arecaidine led to a concentration-dependent inhibition of capsaicin-evoked CGRP release to 39% (EC50=255 nM) and 28% (EC50=847 nM) of control values, respectively. Both responses were blocked by the non-selective mAChR antagonist atropine or the M2 antagonist gallamine. Further, the endogenous ligand ACh produced a bi-phasic response, potentiating evoked CGRP release to 195% of control (EC50= 918nM) and inhibiting evoked CGRP release to 45% of control (EC50=255 microM), effects that were shown to be mediated by nAChRs and mAChRs, respectively. Finally, combined in situ hybridization/immunofluorescence demonstrated that m2 mRNA was present in 20% of trigeminal ganglion neurons between 30 and 60 microm in diameter and that 5-9% of these also expressed CGRP or VR1 immunoreactivity. These results show that activation of peripheral M2 receptors produces antinociception in vivo and the inhibition of nociceptor activity in vitro. While histological analyses at the level of the trigeminal neuronal cell bodies leave open the question of whether the effects of M2 agonists are direct or indirect, these data indicate that primary sensory neuronal M2 receptors may represent a viable peripheral target for the treatment of pain and inflammation.