Slices of rat hypothalamus prelabeled with [3H]-5-hydroxytryptamine ([3H]-5-HT) were superfused and the release of the labeled transmitter was elicited either by electrical stimulation or by fenfluramine. Whereas the electrically stimulated release of [3H]-5-HT was completely abolished by removing calcium from the superfusion medium, the fenfluramine-induced release of [3H]-5-HT was calcium-independent. Methiothepin increased, in a concentration-dependent manner, the [3H]-5-HT release induced by electrical stimulation but had no effect on that elicited by fenfluramine. The 3H-transmitter release elicited by electrical stimulation was inhibited by lysergic acid diethylamide (LSD) in a concentration-dependent manner, but the release induced by fenfluramine was not modified by LSD. The reduction by LSD of [3H]-5-HT overflow elicited by electrical stimulation was antagonized by methiothepin, but unaffected by phentolamine or by sulpiride. Low concentrations (10-1000 nM) of citalopram, a 5-HT uptake inhibitor, antagonized the inhibition by LSD of electrically evoked release of [3H]-5-HT. These concentrations of citalopram did not modify by themselves the overflow of [3H]-5-HT elicited by electrical stimulation. It is concluded that the modulation of [3H]-5-HT release by presynaptic serotonin autoreceptors is not operational when the neurotransmitter is released through a calcium-independent mechanism. The potent presynaptic inhibition by LSD of serotonergic neurotransmission may contribute to the central actions of this drug. The interaction between citalopram and LSD at the level of [3H]-5-HT release does not seem to involve a competitive interaction at the same receptor site. The possibility that neuronal uptake of 5-HT and the presynaptic 5-HT autoreceptor may be linked in a functional manner cannot be excluded.