Objective. The objective of this study was to investigate the expression of adenosine triphosphate P2X3 receptor subunits in rat molar pulp and trigeminal ganglia and their relationship to substance P. Study design. Rat molar pulp and trigeminal ganglia were fixed and sectioned. Double immunostaining with anti-P2X3 and anti-substance P were used to localize P2X3 and substance P expression simultaneously with different stains. Results. P2X3 immunoreactivity (IR) fibers were present in root and coronal pulp. P2X3-IR fibers formed subodontoblastic plexus and advanced into the predentin and dentin. P2X3-IR neurons were localized in small and medium-sized cells in trigeminal ganglia. Colocalization of P2X3 and substance P was not found in either molar pulp or trigeminal ganglia. Conclusions. The results of this study indicated that adenosine triphosphate in pulp tissues may stimulate a subpopulation of non-substance P trigeminal afferent fibers through activation of P2X3 receptors. © 2002, Mosby, Inc.