Electrophysiological techniques were employed to examine the organization of the projections of submucosal neurons in the submucosal plexus of guinea pig ileum. These neurons were activated by focal pressure-pulse application of 5-hydroxytryptamine (5-HT) to single ganglia in submucosal preparations in vitro, and resulting fast excitatory postsynaptic potentials (EPSPs) were recorded intracellularly in S-type neurons. 5-HT-evoked fast EPSPs were blocked by TTX, hexamethonium, and ICS-205-930 (tropisetron). 5-HT was applied either directly to the ganglion containing the neuron recorded intracellularly or to adjacent ganglia positioned at increasing distances on either side of the impaled cell in circumferential or longitudinal orientations. All S-type neurons recorded in this study (n = 103) received nicotinic fast EPSPs from cholinergic neurons when 5-HT was applied directly to the ganglion containing the impaled neuron. Stimulation of adjacent ganglia also evoked nicotinic fast EPSPs, but the number of neurons that received this input decreased as the distance between the stimulus and the impaled cell increased. Maximal projections were 3 mm in the circumferential and orad-to-aborad orientations. There were no significant projections in the aborad-to-orad direction. These findings suggest that S-type neurons in the submucosal plexus are innervated by intrinsic cholinergic neurons that project over relatively short distances and have a distinct orad-to-aborad polarity.