Flat sheet preparations of the mucosa plus submucosa from the guinea-pig ileum were placed in Ussing chambers so that short circuit currrent (I
sc), an index of electrolyte movement across the mucosa, could be measured. In these preparations, 5-hydroxytryptamine (5-HT) increasesI
sc indirectly by stimulating both cholinergic and non-cholinergic secretomotor neurons. The 5-HT3 receptor antagonist, ICS 205–930 (10−13–10−5 M), substantially depressed the secretory response due to 5-HT (10−6 M), but not that produced by direct activation of muscarinic receptors on the mucosal epithelium with carbachol (10−6 M), or by stimulation of secretomotor neurons with substance P (10−8 M) or 1,1-dimethyl-4-phenylpiperazinium (10−5 M). The residual response to 5-HT, after the addition of a maximally effective concentration of ICS 205–930 (10−6 M), was further reduced by hyoscine (10−7M). When that part of the 5-HT response attributable to the release of acetylcholine was blocked by hyoscine (10−7M), ICS 205–930 did not further modify the response to 5-HT. The hyoscine-resistant component was, however, sustantially depressed by tetrodotoxin (3.5 × 10−7 M). The response remaining after ICS 205–930 and hyoscine was not affected by methysergide (2 × 10− 5 M) or cyproheptadine (10−7 M). We conclude that there are ICS 205–930 sensitive 5-HT receptors on cholinergic secretomotor neurons, and ICS 205–930, methysergide, and cyproheptadine insensitive 5-HT receptors on non-cholinergic secretomotor neurons.