Alpha2-adrenoceptor (alpha2-AR)-regulated binding of the labelled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. Under optimal conditions, the potent alpha2-AR agonist, 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), increased the binding of [35S]GTPgammaS up to approximately 1.8 fold, with half-maximal increase at 60 nM and was competitively inhibited by the alpha2-AR antagonist Rauwolscine. The actions of both UK 14304 and Rauwolscine were modulated by monovalent and divalent cation levels, as well as by the concentrations of GDP. [35S]GTPgammaS binding induced by UK 14304 had a Kd value of 4.5 +/- 0.3 nM and a Bmax value of 4.15 +/- 0.40 pmol/mg protein. The rank order of potencies of adrenergic ligands tested in stimulating [3S]GTPgammaS binding and inhibiting forskolin-stimulated c-AMP accumulation was UK 14304> Guanabenz acetate> Oxymetazoline hydrochloride> B-HT 920 dihydrochloride> p-Aminoclonidine hydrochloride> Clonidine hydrochloride. The data presented indicate that enhancement of [35S]GTPgammaS binding by alpha2-AR in human platelet membranes provides a simple functional measure for receptor activation and can be used for determination of potencies and efficacies of ligands at the alpha2-AR.