Musculoskeletal pain conditions, particularly those associated with temporomandibular joint and muscle disorders (TMD) are severely debilitating and affect approximately 12% of the population. Identifying peripheral nociceptive mechanisms underlying mechanical hyperalgesia, a prominent feature of persistent muscle pain, could contribute to the development of new treatment strategies for the management of TMD and other muscle pain conditions. This study provides evidence of functional interactions between ligand-gated channels, P2X3 and TRPV1/TRPA1, in trigeminal sensory neurons, and proposes that these interactions underlie the development of mechanical hyperalgesia. In the masseter muscle, direct P2X3 activation, via the selective agonist αβmeATP, induced a dose- and time-dependent hyperalgesia. Importantly, the αβmeATP-induced hyperalgesia was prevented by pretreatment of the muscle with a TRPV1 antagonist, AMG9810, or the TRPA1 antagonist, AP18. P2X3 was co-expressed with both TRPV1 and TRPA1 in masseter muscle afferents confirming the possibility for intracellular interactions. Moreover, in a subpopulation of P2X3/TRPV1 positive neurons, capsaicin-induced Ca2+ transients were significantly potentiated following P2X3 activation. Inhibition of Ca2+-dependent kinases, PKC and CaMKII, prevented P2X3-mechanical hyperalgesia whereas blockade of Ca2+-independent PKA did not. Finally, activation of P2X induced phosphorylation of serine, but not threonine, residues in TRPV1 in trigeminal sensory neurons. Significant phosphorylation was observed at 15 minutes, the time point at which behavioral hyperalgesia was prominent. Similar data were obtained regarding another nonselective cation channel, the NMDA receptor (NMDAR). Our data propose P2X3 and NMDARs interact with TRPV1 in a facilitatory manner, which could contribute to the peripheral sensitization underlying masseter hyperalgesia. This study offers novel mechanisms by which individual pro-nociceptive ligand gated ion channels form functional complexes in nociceptors. It is also important to further elucidate peripheral anti-nociceptive mechanisms to improve clinical utilization of currently available analgesics and uncover additional therapeutic targets. A side project examined the mechanisms underlying sex differences in the anti-hyperalgesic effects of delta opioid receptors (DORs). This study provides evidence of a sex difference in the potency at DORs that is mediated by differences in the expression of ATP-sensitive potassium channels. Collectively, understanding detailed molecular events that underlie the development of pathological pain conditions could benefit future pharmacotherapies.