Abstract: A new radiolabeled adenosine receptor agonist, 2-[p-(2-carboxyethyl) phenethylamino]-5′-.N-ethylcarboxamidoadenosine (CGS 21680), apparently specific for high-affinity binding sites of the A2 subtype in rat brain, was used to identify and pharmacologically characterize adenosine receptors in human brain. The binding of [3H]CGS 21680, as determined by standard radioligand binding technique in the presence of exogenously added adenosine deaminase, reached equilibrium after 40 min at 25°C. In saturation studies, a single class of high-affinity binding sites with values for KD of 22 ± 0.5 nM and Bmax of 444 ± 63 fmol/mg of protein were observed. Similar binding characteristics were observed regardless of whether rapid filtration or centrifugation was used to separate bound versus free ligand. Of the 14 brain regions examined, [3H]CGS 21680 binding was highest in putamen, followed by globus pallidus and caudate nucleus. The level of [3H]CGS 21680 binding in these areas of basal ganglia was identical to 5′-N-[3H]ethylcarboxamidoadenosine ([3H]NECA) binding in the presence of 50 nM N6-cydopentyladenosine (CPA). The rank order of agonist potencies as determined by a series of competition experiments was NECA > CGS 21680 > 2-chloroadenosine > N6-(R)-phenylisopropyladenosine > N6-cyclohexyladenosine > N6-(S)-phenylisopropyladenosine. This potency order was the same for the binding of [3H]CGS 21680 to rat, and of [3H]NECA in the presence of 50 nM CPA to rat and human, brain membranes. Data from competition studies with labeled and unlabeled CGS 21680 when analyzed by nonlinear regression demon strated the presence of three sites in human basal ganglia with mean values for KD of 16, 1,232, and 15,202 nM, and for Bmax of 305, 3,045, and 30,45 1 fmol/mg of protein. When similar competition experiments were conducted in the absence or presence of 50 nM CPA to determine if the lower-affinity binding components corresponded to labeling of A1 sites, we found that the binding data conformed best to a two-component binding model with KD values of 28 and 2,468 nM and Bmax values of 392 and 3,536 fmol/mg of protein for the high- and low-affinity sites, respectively. For rat striatum, high- and low-affinity sites were observed with kinetic values similar to those of the two higher-affinity sites in human brain. The high-affinity [3H]CGS 21680 binding sites most likely represent A2a sites. The low-affinity sites appear to be composed of a combination of both A1-like and A2b-like adenosine receptors. [3H]CGS 21680 has the potential to become a useful probe in determining the relative importance of A2 sites in mediating the actions of adenosine in structures comprising the basal ganglia and in other discrete regions of mammalian CNS, and may represent the first radioligand available for the study of the previously proposed A2b receptor sites.