The purinoreceptor, P2X3 is a ligand-gated cation channel activated by extracellular ATP. It has been reported that ATP can be released during inflammation and tissue damage, which in turn may activate P2X3 receptors to initiate nociceptive signals. However, little is known about the contribution of P2X3 to the dental pain during pulpal inflammation. Therefore, the purpose of this study was to investigate the expression of P2X3 and its colocalization with TRPV1 to understand the mechanism of pain transmission through P2X3 in the human dental pulp with double labeling immunofluorescence method. In the human dental pulp, intense P2X3 immunoreactivity was observed throughout the coronal and radicular pulp. Of all P2X3-positive fibers examined, 79.4% coexpressed TRPV1. This result suggests that P2X3 along with TRPV1 may be involved in the transmission of pain and potentiation of noxious stimuli during pulpal inflammation.