Experiments were carried out to characterise pharmacologically a neuronal, non-5-HT3, 5-hydroxytryptamine (5-HT) receptor in guinea-pig isolated ascending colon. In preparations pretreated with methysergide (1 microM) and ondansetron (10 microM), 5-HT (0.003-1 microM) produced repeatable concentration-related contractions of guinea-pig ascending colon with an EC50 value of 29 (20-41) nM. The responses to 5-HT could be antagonised substantially by tetrodotoxin (0.3 microM) and atropine (1 microM) indicating a neuronal cholinergically mediated effect. The 5-HT-induced response was mimicked by 5-methoxytryptamine and alpha-methyl-5-HT with equipotent concentration ratios of 26 and 28, respectively. In contrast, 2-methyl-5-HT, 8-OH-DPAT, sumatriptan, phenylbiguanide and 5-hydroxyindalpine had no agonist activity up to 10 microM. The benzamides, metoclopramide, cisapride, R,S-zacopride and renzapride, mimicked the contractile action of 5-HT, acting like partial agonists. ICS205-930 (3 microM) acted as a competitive antagonist against 5-HT and 5-methoxytryptamine with estimated pKB values of 6.4 and 6.7 respectively. ICS205-930 (1 microM) also antagonised responses to R,S-zacopride and renzapride. Ketanserin (1 microM), phenylbiguanide (10 microM) and sulpiride (1 microM) had no effect on responses to 5-HT. We conclude that the pharmacological characteristics of the receptor, which mediates contraction of guinea-pig ascending colon by activation of cholinergic nerves, are consistent with it being of the putative 5-HT4 receptor type.