Conventional intracellular electrophysiological methods were used to study fast synaptic transmission in the myenteric plexus of guinea pig ileum in vitro. Fast excitatory postsynaptic potentials (fEPSPs) were evoked in 98 neurons following single stimuli applied to interganglionic connectives. The nicotinic antagonist hexamethonium (100 microM) reduced fEPSPs by 83% in 37 neurons; these fEPSPs were considered to be cholinergic. In 61 neurons, hexamethonium reduced fEPSPs by 33%; fEPSPs recorded in the presence of hexamethonium were considered to be noncholinergic. Similar data were obtained using the nicotinic antagonist mecamylamine (10 microM) to block fEPSPs. Hexamethonium or mecamylamine completely blocked depolarizations caused by acetylcholine (ACh) applied by ionophoresis. The P2 receptor antagonist suramin (1–300 microM) inhibited noncholinergic fEPSPs in 30 cells; the suramin IC50 was 4 microM. Suramin (100 microM) did not block depolarizations caused by ACh or 5-HT, but suramin blocked depolarizations caused by ATP. Hexamethonium did not block ATP-induced depolarizations. The estimated reversal potential for suramin-sensitive fEPSPs and ATP-induced depolarizations was -25 and -16 mV, respectively. ATP responses were reduced in low-sodium (26 mM) extracellular solution, suggesting that ATP activates a cation channel. These data indicate that in myenteric nerves ATP, in addition to ACh, contributes to fast synaptic transmission.