Intracellular recordings were made from guinea pig submucosal neurons in vitro. Adenosine, 2-[p-(carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine (CGS21680), 2-chloroadenosine (CADO), 5'-N-ethylcarboxamidoadenosine (NECA), R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), N6-cyclohexyladenosine (CHA) and 1-deaza-2-chloro-N6-cyclopentyladenosine (DCCPA) were applied by adding them to the superfusion solution. Adenosine (30 nM to 30 microM) depolarized S-type neurons neurons and this was mimicked by analogs with potency order: CGS21680 = NECA greater than R-PIA greater than CADO greater than adenosine greater than CHA much greater than DCCPA. 8-Cyclopentyltheophylline (CPT) blocked the depolarizing action of CADO or R-PIA; this antagonism was surmountable and the dissociation equilibrium constant (KD) estimated by the Schild method was 295 nM. Synaptic potentials were evoked by focal stimulation of nerve strands running between submucosal ganglia. The nicotinic excitatory postsynaptic potential was reduced by adenosine and analogs with potency order CHA = R-PIA greater than DCCPA = NECA = CADO greater than adenosine much greater than CGS21680; CPT competitively antagonized this effect of CADO or CHA with a KD of 13 nM. The noradrenergic inhibitory postsynaptic potential was also reduced; the potency order was R-PIA greater than CHA = CADO = NECA = DCCPA greater than adenosine much greater than CGS21680; the KD of CPT as an antagonist was 7 nM. It is concluded that adenosine directly depolarizes submucosal neurons by acting at an A2 receptor, and that it inhibits the release of acetylcholine (from intramural nerves) and noradrenaline (from sympathetic nerves) by acting at a presynaptic A1 receptor.