[35S]-GTPgammaS binding has been used to study the function of cloned human 5-HT1D receptor subtypes stably expressed in chinese hamster ovary (CHO) cells. 5-HT stimulated [35S]-GTPgammaS binding to membranes from cells expressing 5-HT1Dalpha or 5-HT1Dbeta receptors. In membranes containing 5-HT1Dbeta receptors, 5-CT and sumatriptan stimulated binding to a similar extent as 5-HT while yohimbine, metergoline and 8-OHDPAT were partial agonists. The order of potency for agonists was 5-CT > 5-HT > metergoline > sumatriptan > yohimbine > 8-OHDPAT. The stimulation of binding by 5-HT in membranes containing 5-HT1Dbeta receptors was potently antagonised by methiothepin (pA2 8.9 +/- 0.1). The overall pharmacological profile for the human 5-HT1Dbeta receptor, defined using [35S]-GTPgammaS binding, agreed well with that reported for inhibition of forskolin-stimulated adenylyl cyclase. In addition, methiothepin and ketanserin inhibited basal [35S]-GTPgammaS binding to membranes containing 5-HT1Dalpha or 5-HT1Dbeta receptors, suggesting that these compounds show negative efficacy at 5-HT1D receptor subtypes. The data show that [35S]-GTPgammaS binding is a suitable method for studying the interaction between cloned human 5-HT1D receptors and G-proteins.