1. The influence of 5-hydroxytryptamine1A (5-HT1A), 5-HT2 and 5-HT3 agonists and antagonists on acetylcholine (ACh) release from the cerebral cortex was studied in freely moving guinea-pigs. 2. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 0.01-1 mg kg-1, s.c.) caused the 5-HT syndrome and dose-dependently increased ACh release. Ru 24969 (1-10 mg kg-1, s.c.) shared the same effects, but it was less potent. (-)-Propranolol (5 mg kg-1) and metitepine (2 mg kg-1) prevented these behavioural and neurochemical responses. 3. (+/-)-1(4-Iodo-2,5-dimethoxyphenyl)2-aminopropane (DOI) up to 2 mg kg-1 did not modify ACh release and ketanserin (0.5 mg kg-1) was ineffective on 5-HT-induced changes of ACh outflow. 4. 2-Methyl-5-HT (500 micrograms, i.c.v.) and 5-HT (500 micrograms, i.c.v.) plus metitepine (2 mg kg-1, s.c.) inhibited the gross behaviour and ACh release. ICS 205-930 (0.5 mg kg-1) prevented these responses. 5. 2-Methyl-5-HT, up to 10 mumols 1(-1), and 8-OH-DPAT, up to 0.1 mumols 1(-1), (like 5-HT) did not change [3H]-choline efflux from cerebral cortex slices. 6. These results suggest that exogenous 5-HT and related selective agonists modulate guinea-pig cortical cholinergic structures through 5-HT1A and 5-HT3 receptors. The stimulation of 5-HT1A autoreceptors may lead to disinhibition of the cholinergic cells, tonically inhibited by the tryptaminergic control. Conversely, the stimulation of 5-HT3 receptors inhibits ACh release, possibly through an interneurone. No direct 5-HT modulation of the cholinergic nerve endings was found.