Acetylcholine (ACh) is a major excitatory neurotransmitter in the myenteric plexus, and it regulates its own release acting via muscarinic autoreceptors. Adenosine released from stimulated myenteric neurons modulates ACh release preferentially via facilitatory A(2A) receptors. In this study, we investigated how muscarinic and adenosine receptors interplay to regulate ACh from the longitudinal muscle-myenteric plexus of the rat ileum. Blockade of the muscarinic M(2) receptor with 11-[[2-1[(diethylamino) methyl-1-piperidinyl]- acetyl]]-5,11-dihydro-6H-pyrido [2,3-b][1,4] benzodiazepine-6-one (AF-DX 116), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and atropine facilitated [3H]ACh release evoked by short stimulation trains (5 Hz, 200 pulses). Prolonging stimulus train length (>750 pulses) shifted muscarinic autoinhibition towards facilitatory M(3) receptors activation, as predicted by blockade with J104129 (a selective M(3) antagonist), 4-DAMP and atropine, whereas the selective M(2) antagonist, AF-DX 116, was without of effect. Blockade of A(2A) receptors with ZM 241385, inhibition of adenosine transport with dipyridamole, and inhibition of ecto-5'-nucleotidase with concanavalin A, all attenuated release inhibition caused by 4-DAMP. J104129 and 4-DAMP, but not AF-DX 116, decreased ( approximately 60%) evoked adenosine outflow (5 Hz, 3000 pulses). Oxotremorine (300 micromol L(-1)) facilitated the release of [3H]ACh (34 +/- 4%, n = 5) and adenosine (57 +/- 3%, n = 6) from stimulated myenteric neurons. 4-DAMP, dipyridamole and concanavalin A prevented oxotremorine-induced facilitation. ZM 241385 blocked oxotremorine facilitation of [3H]ACh release, but kept adenosine outflow unchanged. Thus, ACh modulates its own release from myenteric neurons by activating inhibitory M(2) and facilitatory M(3) autoreceptors. While the M(2) inhibition is prevalent during brief stimulation periods, muscarinic M(3) facilitation is highlighted during sustained nerve activity as it depends on extracellular adenosine accumulation leading to activation of facilitatory A(2A) receptors.