The coexistence of both inhibitory A1 and facilitatory A2 adenosine receptors in the rat myenteric plexus prompted the question of how adenosine activates each receptor subtype to regulate cholinergic neurotransmission.Exogenously applied adenosine (0.3–300 μM) decreased electrically evoked [3H]acetylcholine ([3H]ACh) release. Blocking A1 receptors with 1,3-dipropyl-8-cyclopentylxanthine (10 nM) transformed the inhibitory action of adenosine into a facilitatory effect. Adenosine-induced inhibition was mimicked by the A1 receptor agonist R-N6-phenylisopropyladenosine (0.3 μM), but the A2A agonist CGS 21680C (0.003 μM) produced a contrasting facilitatory effect.Increasing endogenous adenosine levels, by the addition of (1) the adenosine precursor AMP (30–100 μM), (2) the adenosine kinase inhibitor 5′-iodotubercidin (10 μM) or (3) inhibitors of adenosine uptake (dipyridamole, 0.5 μM) and of deamination (erythro-9(2-hydroxy-3-nonyl)adenine, 50 μM), enhanced electrically evoked [3H]ACh release (5 Hz for 40 s). Release facilitation was prevented by adenosine deaminase (ADA, 0.5 U ml−1) and by the A2A receptor antagonist ZM 241385 (50 nM); these compounds decreased [3H]ACh release by 31±6% (n=7) and 37±10% (n=6), respectively.Although inhibition of ecto-5′-nucleotidase by α,β-methylene ADP (200 μM) or by concanavalin A (0.1 mg ml−1) attenuated endogenous adenosine formation from AMP, analysed by HPLC, the corresponding reduction in [3H]ACh release only became evident when stimulation of the myenteric plexus was prolonged to over 250 s.In summary, we found that endogenously generated adenosine plays a predominantly tonic facilitatory effect mediated by prejunctional A2A receptors. Extracellular deamination and cellular uptake may restrict endogenous adenosine actions to the neuro-effector region near the release/production sites.