The effects of various cholinergic agonists and antagonists on the spontaneous release of newly synthetized 3H-5-HT were examined in rat hypothalamic slices. 3H-5-HT was measured in incubating medium at the end of a 30 min incubation carried out with L-3H-tryptophan in the presence of the various drugs tested. ACh (10(-5) M) in the presence of eserine (2 X 10(-4) M), and carbachol (10(-5) M) stimulated the release of 3H-5-HT. In contrast, oxotremorine (10(-5) M) reduced the 3H-amine release. The effect of carbachol was blocked by two nicotinic blockers, mecamylamine (10(-6) M) and d-tubocurarine (10(-6) M). It was not reduced by the muscarinic antagonists, atropine (10(-6) M) and scopolamine (10(-6) M). In fact, each of two antagonists added alone to the incubating medium enhanced 3H-5-HT release. The scopolamine (10(-6) M) stimulating effect on 3H-5-HT release was suppressed by d-tubocurarine (10(-6) M). Finally, the inhibiting effect of oxotremorine on 3H-5-HT release was not prevented by d-tubocurarine (10(-6) M) but was in the presence of atropine (10(-6) M) or scopolamine (10(-6) M). In the concentrations used in the release study, the cholinergic agonists and antagonists had no effect on the total formation of 3H-5-HT and 3H-5-HIAA from L-3H-tryptophan and on the accumulation of L-3H-tryptophan in tissues. In these concentrations, except for eserine, they did not affect the uptake of exogenous 3H-5-HT in hypothalamic synaptosomes (P2 fraction). These results suggest that cholinergic receptors of the muscarinic and nicotinic type are involved in the control of 3H-5-HT release; since the stimulation of the muscarinic and nicotonic cholinergic receptors resulted in an inhibition and an activation of 3H-5-HT release, respectively. As in the case of peripheral noradrenergic and central dopaminergic neurons the cholinergic receptors could be localized on serotoninergic terminals.