Activation of P2X receptors by a Ca²⁺- and SNARE protein-dependent release of ATP was measured using patch clamp recordings from dissociated guinea pig stellate neurons. Asynchronous transient inward currents (ASTICs) were activated by depolarization or treatment with the Ca²⁺ ionophore ionomycin (1.5 & 3 µM). During superfusion with a HEPES-buffered salt solution containing 2.5 mM Ca²⁺, depolarizing voltage steps (-60 to 0 mV, 500 ms) evoked ASTICs on the decaying phase of a larger transient inward current. Equimolar substitution of Ba²⁺ for Ca²⁺ augmented the post-depolarization frequency of ASTICs while eliminating the larger transient current. Perfusion with an ionomycin containing solution elicited a sustained activation of ASTICs, allowing quantitative analysis over a range of holding potentials. Under these conditions, increasing extracellular [Ca²⁺] to 5 mM increased ASTIC frequency while no events were observed following replacement of Ca²⁺ with Mg²⁺ demonstrating a Ca²⁺ requirement. ASTICs were Na⁺ dependent, inwardly rectifying and reversed near 0 mV. Treatment with the nonselective purinergic receptor antagonist PPADS (10 µM) blocked all events under both conditions while the ganglionic nicotinic antagonist hexamethonium (100 µM & 1 mM) had no effect. PPADS also blocked the macroscopic inward current evoked by exogenously applied ATP (300 µM). The presence of botulinum neurotoxin E (BoNT/E) in the whole cell recording electrode significantly attenuated the ionomycin induced ASTIC activity while phorbol ester treatment potentiated this activity. These results suggest ASTICs are mediated by vesicular release of ATP and activation of P2X receptors.