The lack of a radiolabeled selective A₃ adenosine receptor antagonist is a major drawback for an adequate characterization of this receptor subtype. This paper describes the pharmacological and biochemical characterization of the tritiated form of a new potent A₃ adenosine receptor antagonist, the pyrazolo triazolo pyrimidine derivative [³H]5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e] -1,2,4- triazolo[1,5-c]pyrimidine ([³H]MRE 3008F20). [³H]MRE 3008F20 bound specifically to the human adenosine A₃ receptor expressed in CHO cells (hA₃CHO), and saturation analysis revealed a single high affinity binding site, K _D = 0.80 ± 0.06 nM, with a B _max = 300 ± 33 fmol/mg protein. This new ligand displayed high selectivity (1294-, 165-, and 2471-fold) in binding assay to human A₃ versus A₁, A_2A, and A_2B receptors, respectively, and binds to the rat A₃ receptors with a K _i > 10 μM. The pharmacological profile of [³H]MRE 3008F20 binding to hA₃CHO cells was evaluated using known adenosine receptor agonists and antagonists with a rank order of potency consistent with that typically found for interactions with the A₃ adenosine receptors. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency identical with that observed in binding experiments. Thermodynamic data indicated that [³H]MRE 3008F20 binding to hA₃CHO is entropy- and enthalpy-driven in agreement with the typical behavior of other adenosine antagonists to A₁ and A_2A receptors. These results show that [³H]MRE 3008F20 is the first antagonist radioligand with high affinity and selectivity for the human A₃ adenosine receptor and may be used to investigate the physiopathological role of A₃ adenosine receptors.