1. 5-Hydroxytryptamine (5-HT) stimulated an increase in short-circuit current (SCC) in rat isolated colonic mucosa with an EC50 value of approximately 4 microM. The purpose of the present study was to investigate the 5-HT receptor mechanism(s) involved in this response. 2. The relatively selective 5-HT receptor agonists 5-carboxamidotryptamine (5-CT) and alpha-methyl-5-HT stimulated SCC and were 6 to 8 times less potent than 5-HT. 2-Methyl-5-HT was inactive both as an agonist and an antagonist. 3. The following compounds produced no significant inhibition of the SCC response to 5-HT: ketanserin (1 microM), methysergide (1 microM), methiothepin (0.3 microM), GR38032F (0.3 microM), tetrodotoxin (0.3 microM) and sulpiride (1 microM). 4. Both metoclopramide (3 and 10 microM) and cisapride (0.1 and 1 microM) inhibited the SCC responses to 5-HT in a concentration-related manner, and the higher doses similarly inhibited the responses to 5-CT. With both agonists the inhibitory effects of metoclopramide and cisapride were insurmountable. However, these inhibitory actions appeared to be selective since neither metoclopramide nor cisapride affected the basal SCC or the SCC response to prostaglandin E2. 5. The SCC responses to 5-HT and 5-methoxytryptamine were selectively inhibited by ICS205-930 at 3 microM, and respective pKB values of 6.0 and 6.6 were calculated. 6. It is concluded that 5-HT stimulates an SCC response in rat colon via a receptor mechanism that cannot be clearly identified as 5-HT1-like, 5-HT2 or 5-HT3. This receptor is selectively antagonized by ICS 205-930 and by the benzamides, metoclopramide and cisapride. The 5-HT receptor in rat colon therefore exhibits some of the properties associated with the so-called 5-HT4 receptor.