The present study was designed to characterize an "atypical" 5-hydroxytryptamine (5-HT) receptor mediating relaxation of the rat oesophageal tunica muscularis mucosae. All experiments were performed under equilibrium conditions, using pargyline to inhibit the oxidative deamination of indoleamines, and cocaine and corticosterone to inhibit neuronal and extraneuronal uptake. Under these conditions 5-HT (0.3-1000 nmol/l) produced a concentration-dependent relaxation of carbachol-induced tension. The concentration-effect curve to 5-HT was unaffected by potent antagonists for 5-HT1, 5-HT2, 5-HT3 and so called 5-HT1P receptors (metergoline, methysergide, ketanserin, ondansetron, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide), but was antagonized competitively by ICS 205-930 (pA2 = 6.7). Responses to 5-HT were mimicked by other indoleamines and substituted benzamides with the following order of potency: 5-HT greater than or equal to 5-methoxytryptamine greater than cisapride = alpha-methyl-5-HT = (S)-zacopride = renzapride greater than (RS)-zacopride greater than 5-carboxamido-tryptamine = metoclopramide = (R)-zacopride greater than tryptamine greater than 2-methyl-5-HT. ICS 205-930 afforded similar pA2 values (6.0-6.7) against each agonist, indicating a common site of action. Concentration-effect curves to 5-HT were not affected by tetrodotoxin or indomethacin, suggesting that 5-HT-induced relaxation of the tunica muscularis mucosae was mediated via a post-junctional receptor, independent of endogenous prostanoids. The pharmacological profile of the 5-HT receptor in the rat oesophageal tunica muscularis mucosae correlates well with the 5-HT4 receptor characterized recently in both the CNS and gastro-intestinal tract.