Extracellular ATP, an essential pain mediator, is received by cell-surface ionotropic P2X and/or metabotropic P2Y receptors. Although the contribution of P2X₃ and/or P2X(2/3) receptors toward the pain mechanism is well described in trigeminal ganglion neurons, the expression of other subtypes of P2X receptor remains to be clarified. We examined expression of P2X receptor mRNA and measured intracellular free Ca²⁺ concentration ([Ca²⁺]i) by the activation of these receptors by fura-2 fluorescence in primary cultured rat trigeminal ganglion neurons. Real-time reverse transcription-PCR analysis revealed mRNA expression of P2X receptor subtype P2X₁, P2X₃, and P2X₄ in trigeminal ganglion neurons. In the presence of extracellular Ca²⁺, the application of P2X receptors agonists, ATP, α,β-methylene ATP or β,γ-methylene ATP induced Ca²⁺ influx significantly. The ATP-induced increase in [Ca²⁺]i was inhibited by a series of selective antagonists for P2X₁, P2X₃, or P2X₄ receptors. These results indicate that trigeminal ganglion neurons functionally express P2X₁, P2X₃, and P2X₄ receptors and that these receptors are involved in the mediation of not only nociceptive but also neuropathic pain in the orofacial area.