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The A_2A adenosine receptor (A_2A‐AR) transcript and radioligand binding sites have a distinct distribution in rat brain, restricted primarily to the striatum, nucleus accumbens and olfactory tubercles. We describe here the use of purified recombinant human A_2A‐ARs to generate a monoclonal antibody that has been used to better resolve the distribution of A_2A‐ARs in rat brain. The antibody can detect 1 ng of purified recombinant receptor by Western blotting and is potent (EC₅₀ = 0.62 μg/ ml) and highly selective for the A_2A‐AR subtype. By Western blotting, the apparent molecular mass of recombinant and rat striatal receptors shifts upon deglycosylation from 43–48 to 42 kilodaltons. Analyses of chimeric A₁/ A_2A‐ARs and synthesis of a blocking peptide pinpointed the epitope (SQPLPGER) of the antibody to the center of the third intracellular loop of the receptor. Incubation of rat striatal membranes with antibody …