The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K+ were determined in superfused synaptosomes and slices, preincubated with [3H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [3H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT1A and 5-HT2C sites) to guinea-pig cortical synaptosomes and membranes. 5-HT receptor agonists inhibited the K(+)-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl] -1H-indole-3-yl] ethylamine (L-694,247) > 5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) > or = 5-HT > sumatriptan > or = 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT1D alpha and 5-HT1D beta receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA2: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT1D alpha, but not the 5-HT1D beta, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [3H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [3H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin > 5-CT > 5-methoxytryptamine > 5-HT > or = sumatriptan > or = oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [3H]5-HT binding to synaptosomes and membranes. In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [3H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT1D beta-like.