²⁺]_i We characterized ATP-induced changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane current in cultured rat myenteric neurons using ratiometric Ca²⁺ imaging with fura-2 and the whole-cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca²⁺]_i responses to high-K and nicotine which occurred only in cells positive for neuron specific PGP 9.5-immunoreactivity. ATP evoked dose-dependent increase of [Ca²⁺]_i, that was greatly decreased by the removal of extracellular Ca²⁺ ([Ca²⁺]_o). The amplitude of the [Ca²⁺]_i response to ATP was reduced by half in the presence of voltage-dependent Ca²⁺ channel blockers. In [Ca²⁺]_o-free solution, ATP produced a small transient rise in [Ca²⁺]_i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of [Na⁺]_o. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca²⁺]_i was ATPATPSCTP2methylthioATP>benzoylbenzoylATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca²⁺ and Mg²⁺ suppressed the ATP-induced current, and Zn²⁺, Cu²⁺ and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X₂ receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X₂ receptors, resulting in a [Ca²⁺]_i increase dependent on [Ca²⁺]_o in rat myenteric neurons. A small part of the ATP-induced [Ca²⁺]_i increase may be also mediated via a P2Y receptor-related mechanism.