The binding characteristics of the selective adenosine A2 agonist [3H]CGS 21680 ([3H]2-[p-(2-carboxyethyl)-phenethyl-amino]-5'-N- ethylcarboxamidoadenosine) were determined in human platelet membranes. Specific binding was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 1.4 microM and 5.9 pmol/mg of protein, respectively. Adenosine receptor agonists and antagonists competed for the binding of [3H]CGS 21680 (50 or 200 nM) to human platelet membranes showing a rank order of potency consistent with that typically found for interactions at the adenosine A2 receptor. Adenylate cyclase stimulation and platelet aggregation inhibition induced by adenosine agonists exhibited a rank order of potency close to that observed in binding experiments. However, the adenosine A1 receptor agonists, R- and S-N6-(2-phenylisopropyl)adenosine, (R-PIA) and (S-PIA), N6-cyclohexyladenosine (CHA) and 2-chloro-N6-cyclopentyladenosine (CCPA), which stimulate adenylate cyclase and inhibit platelet aggregation in the low microM range, displaced [3H]CGS 21680 only in the high microM range. In conclusion, we have found that [3H]CGS 21680, which is widely used as a specific A2 agonist in binding studies on brain tissues, is not appropriate for the characterization of the human platelet adenosine A2 receptor.