The serotonin (5-HT) releaser d-fenfluramine and its active metabolite d-norfenfluramine, or the 5-HT-uptake inhibitor citalopram, by increasing synaptic 5-HT availability, facilitated in vivo release of acetylcholine (ACh) from dorsal hippocampi of freely moving rats as determined by the microdialysis technique. The effects of d-norfenfluramine (7.5 mg/kg i.p.) and citalopram (10 microM, applied by reverse dialysis) were prevented by a 14-day chemical lesion of the raphe nuclei, suggesting mediation by the 5-HT system in the cholinergic action of the drugs. The increase in extracellular ACh content induced by d-norfenfluramine (5 mg/kg i.p.) was antagonized by the 5-HT3 receptor antagonists tropisetron (0.5 mg/kg i.p.) and DAU 6215 (60 micrograms/kg i.p.), but not by the mixed 5-HT1 and 5-HT2 receptor antagonist metergoline (2 mg/kg s.c.). In accordance with an involvement of the 5-HT3 receptor in the ACh facilitation induced by d-norfenfluramine is the finding that the selective 5-HT3 receptor agonist 2-methylserotonin (250 micrograms i.c.v., or 10 microM applied by reverse dialysis) raised ACh release. The effect of the intracerebroventricular drug was prevented by the 5-HT3 antagonists DAU 6215 (60 micrograms/kg i.p.) and ondansetron (60 micrograms/kg s.c.). These antagonists by themselves did not modify the basal ACh release, indicating that 5-HT does not tonically activate the 5-HT3 receptors involved. In conclusion, the overall regulatory control exerted by 5-HT in vivo is to facilitate hippocampal ACh release. This is mediated by 5-HT3 receptors probably located in the dorsal hippocampi.