1. Neuronal 5-hydroxytryptamine (5-HT) receptors mediating contraction of guinea-pig ileal segments have been characterized in vitro by the use of methysergide to block 5-HT1-like and 5-HT2 receptors. Concentration-response curves to 5-HT were biphasic (first phase, defined as those responses occurring between 1 nM and 0.32 microM 5-HT, -log EC50 = 7.15 +/- 0.08; second phase, defined as these responses occurring between 0.32 microM and 32 microM 5-HT, -log EC50 = 5.32 +/- 0.03) but monophasic to 5-methoxytryptamine (-log EC50 = 7.0 +/- 0.08) and 2 methyl 5-HT (-log EC50 = 5.2 +/- 0.13). The maximal response of the first phase to 5-HT and the maximal response to 5-methoxytryptamine were 30 +/- 4% and 35 +/- 5% respectively of the maximum response to the second phase of the 5-HT concentration-effect curve (set at 100%). In contrast, the maximal response to 2-methyl-5-HT equalled that obtained with 5-HT (second phase). 2. The responses comprising the second phase of the concentration-effect curve to 5-HT were antagonized by 1 microM ICS 205-930, ondansetron, granisetron, quipazine, N-methyl-quipazine and (R,S)-zacopride and the following pKB values, with 5-HT as the agonist, were obtained at the 5-HT3 receptor: ICS 205-930 7.61 +/- 0.05, ondansetron 6.90 +/- 0.04, granisetron 7.90 +/- 0.04, (S)-zacopride 8.11 +/- 0.06, (R,S)-zacopride 7.64 +/- 0.11, and (R)-zacopride 7.27 +/- 0.06. 3. Under conditions of 5-HT1-like, 5-HT2 and 5-HT3 receptor blockade, the following rank order of agonism was observed: 5-HT > 5-methoxytryptamine = renzapride > (S)-zacopride > (R,S-zacopride > 5-carboxamidotryptamine > BRL 24682 > (R-zacopride > metoclopramide > 2-methyl-5-HT > sulpiride. 8-Dihydroxydiphenylaminotetralin (8-OHDPAT), GR 43175, N,N-dipropyl-5-carboxamidotryptamine, ondansetron, ICS 205-930, granisetron, quipazine and N-methyl-quipazine were inactive as agonists and antagonists. Relative to 5-HT, (R,S)-zacopride acted as a partial agonist (intrinsic activity, alpha = 0.80; -log EC50 = 6.3 + 0.12; -log KA = 6.1 + 0.03) as did (R)-zacopride (alpha = 0.4, -log EC,0 5.7 + 0.08, -log KA = 5.5 + 0.11). (S)-zacopride acted as a full agonist (-log EC,0 = 6.9 + 0.03). ICS 205-930 (3 microM) antagonized competitively responses to 5-HT, 5 methoxytryptamine, (RS)- and (S)- zacopride and 5-carboxamidotryptamine yielding -log KB estimates ranging from 6.1-6.5. 4. It is concluded that two different 5-HT receptors mediate excitatory neuronal responses in the guineapig ileum. 5-HT3 receptors mediate the second phase of the biphasic concentration-response curve, whereas a receptor with properties distinct from the 5-HT1-like, 5-HT2 and 5-HT3 subtypes mediates the initial phase of the concentration-response curve. This receptor, which exhibits a close similarity to the 5-HT4 subtype is: (1) stimulated by 5-methoxytryptamine but not 2-methyl-5-HT; (2) stimulated selectively by certain substituted benzamides; (3) recognizes the optical isomers of zacopride and (4) is blocked by relatively high concentrations ICS 205-930 (pKB = 6.0-6.5) but not ondansetron, granisetron, quipazine or N-methyl-quipazine.