We have previously shown that a non-classical 5-hydroxytryptamine (5-HT₄) receptor mediates the stimulation of adenylate cyclase activity in mouse embryo colliculi neurons in primary culture. The pharmacological characteristics of this receptor exclude the possibility that it belongs to the known 5-HT₁, 5-HT₂ or 5-HT₃ receptor types. Here we report that this 5-HT receptor can be stimulated by 4-amino-5-chloro-2-methoxy substituted benzamide derivatives. All these compounds have been reported to be potent stimulants of gastrointestinal motility and some of them are 5-HT₃ receptor antagonists. The rank order of potency of these substituted benzamide derivatives in stimulating cAMP formation was: cisapride > BRL 24924 > 5-HT > zacopride > BRL 20627 > metoclopramide. The non-additivity of benzamide and 5-HT activities suggests that 5-HT and the substituted benzamide derivatives act on the same receptor. Only ICS 205930, a recognized 5-HT₃ receptor antagonist, competitively antagonized the stimulatory effect of cisapride, zacopride and BRL 24924. However, its pK _i (6–6.3) for this new receptor was very different from its pK _i for 5-HT₃ receptors (pK _i = 8 –10). Other selective 5-HT₃ receptor antagonists with an indole group (BRL 43694 and GR 38032F), with a benzoate group (cocaïne, MDL 72222) or with a piperazine group (quipazine) were ineffective in reversing the stimulatory effect of benzamide derivatives. Exposure of neuronal cells to potent agonists at this receptor such as BRL 24924 rapidly reduces its capacity to stimulate cAMP production. For example, a preincubation of 10 min with BRL 24924 (100 μmol/l) reduced by 42% the ability of 5-HT to stimulate cAMP production. Cross-desensitization occurs between the effects of 5-HT and benzamides. The unique pharmacology of these nonclassical 5-HT receptors that we propose to call 5-HT₄ is very close and even identical to the pharmacology of the high affinity 5-HT receptors involved in the indirect stimulation of smooth muscle in the guinea pig ileum. These receptors are different from the 5-HT₃ receptors also present in guinea pig ileum.