The neurotransmitter(s) underlying NOS-independent neural inhibition in the IAS is still uncertain. The present study investigated the role of purinergic transmission. Contractile and electrical responses to electrical field stimulation of nerves (0.1-5 Hz for 10-60 sec) were recorded in strips of mouse IAS. A single stimulus generated a 28 mV fast inhibitory junction potential (F-IJP) and relaxation. The NOS inhibitor L-NNA reduced the F-IJP duration by 20%. Repetitive stimulation at 2.5-5 Hz caused a more sustained IJP and sustained relaxation. L-NNA reduced relaxation at 1 Hz and the sustained IJP at 2.5-5 Hz. All other experiments were carried out in the presence of NOS blockade. IJPs and relaxation were significantly reduced by the P2 receptor antagonists PPADS (100µM), by desensitization of P2Y receptors with ADPS (10µM) and by the selective P2Y1 receptor blocker MRS2179 (10µM). Relaxation and IJPs were also significantly reduced by the K⁺ channel blocker apamin (1µM). Removal of extracellular potassium (K_o) increased IJP amplitude to 205% of control whereas return of K_o 30 minutes later hyperpolarized cells by 19 mV and reduced IJP amplitude to 50% of control. Exogenous ATP (3mM) relaxed muscles in the presence of TTX (1µM) and hyperpolarized cells by 15 mV. In conclusion, these data suggest that purinergic transmission significantly contributes to NOS-independent neural inhibition in the mouse IAS. P2Y1 receptors as well as at least one other P2 receptor subtype contribute to this pathway. Purinergic receptors activate apamin sensitive K⁺ channels as well as other apamin-insensitive conductances leading to hyperpolarization and relaxation.