Compounds able to discriminate functionally between the closely related cloned human 5-HT1D alpha and 5-HT1D beta receptor subtypes have not been reported previously. In [3H]5-HT competition assays, the classical 5-HT2A receptor antagonists, ritanserin and ketanserin, displayed moderate affinity (pKi = 7.30 and 7.17, respectively) and marked selectivity (22- and 71-fold, respectively) for the recombinant human 5-HT1D alpha subtype relative to the 5-HT1D beta receptor. In contrast, the nonselective 5-HT1/2 receptor antagonist, methiothepin, exhibited similar binding affinities (pKi = 7.64-8.01) for both recombinant 5-HT1D subtypes. The antagonistic properties of these compounds were evaluated for their ability to block 5-HT-induced inhibition of forskolin-stimulated cAMP accumulation in intact cells stably expressing either 5-HT1D alpha or 5-HT1D beta receptors. All three compounds behaved as antagonists devoid of intrinsic activity in the functional assays. The apparent pKb values determined in functional assays closely matched their pKi values obtained in binding assays. Since ketanserin exhibits significant selectivity for the human 5-HT1D alpha receptor, this antagonist can be used as a pharmacological tool to discriminate between 5-HT1D alpha and 5-HT1D beta receptor-mediated responses in human tissues.