Alpha 2-Adrenergic receptor-mediated inhibition of adenylate cyclase requires the guanine nucleotide-binding protein, Ni. This protein may also be required for stabilization of high-affinity alpha 2-adrenergic agonist binding. Human platelet membranes treated under alkaline conditions (pH 11.5) exhibited a selective loss of high-affinity agonist binding as measured by p-[3H]aminoclonidine and [3H]UK 14,304. Binding of the antagonist [3H]yohimbine was largely unaffected with retention of greater than 60% of control binding sites. Ni, determined by pertussis toxin-catalyzed [32P]ADP-ribosylation of cholate extracts from alkaline-treated membranes, was also markedly reduced. The parallel loss of alpha 2-agonist binding and Ni provides additional evidence that Ni is required for alpha 2-adrenergic agonist binding.