Human cerebral cortical synaptosomes were used to determine the 5-hydroxytryptamine (5-HT) receptor subtype to which the inhibitory presynaptic 5-HT autoreceptor belongs. The synaptosomes preincubated with [3H]5-HT were superfused and tritium overflow was stimulated by high K+. The K(+)-evoked tritium overflow, which was Ca(2+)-dependent but tetrodotoxin-resistant, was concentration-dependently inhibited by the nonselective 5-HT 1D alpha/1D beta receptor agonist, 5-carboxamidotryptamine. Ketanserin at a concentration which should block the 5-HT 1D alpha but not the 5-HT 1D beta receptor failed to antagonize the inhibitory effect of 5-carboxamidotryptamine. In contrast, the nonselective 5-HT 1D alpha/1D beta receptor antagonist, methiothepin, at a concentration which should block both the 5-HT 1D alpha and the 5-HT 1D beta receptor abolished the effect of 5-carboxamidotryptamine. It is concluded that the presynaptic 5-HT autoreceptor, which has previously been classified as 5-HT 1D, belongs to the 5-HT1D beta subtype.