Intracellular recording methods were used to investigate the interactions between serotonin (5-HT) and cisapride on myenteric neurons of guinea-pig small intestine. Serotonin had three actions on the neurons. One was a slowly rising depolarization associated with increased input resistance and discharge of spikes that lasted six or more times longer than the duration of the 5-HT application. The second action was a transient depolarization associated with decreased input resistance and brief discharge of spikes. This response desensitized quickly and could be evoked only at intervals of 2 to 3 min. The third action of 5-HT was presynaptic inhibition of acetylcholine release at nicotinic synapses. Cisapride reduced or abolished both the prolonged and transient responses to 5-HT. The threshold concentration for reduction of the responses was 0.1 microM and the responses were abolished at 1.0 to 10 microM. Cisapride suppressed stimulus-evoked slow excitatory postsynaptic potentials (EPSPs) in the same cells for which cisapride blocked the prolonged responses to 5-HT. There were no effects of cisapride on resting electrical behavior or spike generation. Cisapride reduced the amplitude of fast cholinergic EPSPs, suggesting that it behaved as an agonist at the presynaptic serotonergic receptors.