Alpha-2 adrenergic receptor isotypes have been proposed to explain several pharmacologic differences between rodent and nonrodent species. In support of this hypothesis, we found that the differences in the pharmacologic properties of rat cerebral cortex and human platelet alpha-2 adrenergic receptors were not due to 1) differential proteolysis of the receptor, 2) degradation of the ligand, 3) the detection of different affinity states or 4) the presence of different quantities of various affinity modulators. In an effort to test the hypothesis further we have characterized these prototype isoceptors in soluble form. Soluble receptors from both species showed the appropriate rank order of potencies for various adrenergic agonists and antagonists expected for an alpha-2 adrenergic receptor. The KD values for soluble human platelet and rat cerebral cortical alpha-2 receptors were 4.2 and 5.9 nM, respectively. The species differences in the affinities of prazosin and oxymetazoline were retained upon solubilization. Both soluble receptors were insensitive to modulation by guanine nucleotides, indicating uncoupling from the inhibitory regulatory subunit Ni. Sucrose density gradient centrifugation of soluble receptors did not indicate any significant molecular size differences.