Injury of the tooth pulp is often excruciatingly painful and yet the receptors and neural circuit mechanisms that transmit this form of pain remain poorly defined in both clinical human and preclinical rodent models. The role of purinergic pathways are of particular interest and potential relevance in dental inflammation and pain. This study examined the involvement of purinergic receptors in human dental inflammatory pain by comparing their distribution in healthy pulps compared to symptomatic and asymptomatic inflamed pulps, and then testing for sex differences in expression. Ionotropic P2X2R and P2X3R were selected for their implication in pain; whereas ecto-nucleotidase NTPDase1 (CD39) reflects extracellular ATP concentration. We found P2X3R and P2X2R, as well as CD39, to colocalize with protein gene product 9.5 (PGP9.5)-positive nerves in control tissues. P2X3R was additionally found on odontoblasts, and CD39 on other non-neuronal structures. Both immunohistochemistry and immunoblots demonstrated that all three proteins were significantly increased in symptomatic pulpitis, suggesting both receptors and agonist ATP were elevated with increased pain. Increased expression of P2X3 and CD39 were more frequently observed in women than men. Our findings support a role for increased purinergic signaling in humans with inflammatory dental pain, with preference in women. This differential response suggests purinergic signaling may contribute to sex differences in pain. To further study signaling pathways of painful pulpitis in the preclinical model, quantifiable behavioral models, namely the Mouse Grimace Scale (MGS) and Facial von Frey assay, were evaluated for their validity in assessing dental pulp pain in mice. We found that unilateral pulp exposure injury to the first maxillary molar in mice resulted in significant increase in MGS from 24 h onward, indicating presence of spontaneous pain. Mechanical allodynia, assessed by the Facial von Frey assay, progressed more gradually with significant increases on both sides of the face on day 4, and unwillingness to tolerate filaments above 0.6 g by day 6 post-injury. This work demonstrates that we have clear, easily identifiable behavioral readouts for trigeminal nociception in the mouse following dental injury.