The potent alpha 2-adrenergic receptor antagonist 3H-yohimbine was used to characterize alpha-adrenergic receptors in human platelet membranes. Binding of 3H-yohimbine was at 25 degrees rapid (t 1/2 = 3 min) readily reversible (t 1/2 = 5.5 min), saturable with 221 +/- 38.9 fmoles bound/mg protein (N = 10) and of high affinity (KD = 1.97 nM). Inhibition of binding by alpha-adrenergic antagonists showed monophasic displacement curves with Hill-coefficients of approximately 1.0. The rank order of potency was: rauwolscine greater than or equal to yohimbine greater than phentolamine greater than phenoxybenzamine greater than AR-C 239 greater than or equal to corynanthine greater than prazosin, indicating that the alpha-adrenergic receptor in human platelets is of the alpha 2-subtype. On the contrary, agonist (clonidine, guanfacine, alpha-methyl-noradrenaline, noradrenaline and adrenaline) displacement curves were shallow with Hill-coefficients of approximately 0.7. Non-linear regression analysis showed that agonists bind to two affinity states of the alpha 2-adrenergic receptor, a high and a low affinity state. In the presence of GTP (10(-4) M) agonist concentration-inhibition curves were shifted to the right to lower affinities and Hill-coefficients increased up to 1.0 K1-values for inhibition of binding in the presence of GTP were in the same range as those for low affinity state in the absence of GTP. It is concluded that GTP regulates binding of alpha 2-adrenergic agonists at the human alpha 2-adrenergic receptor.