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 reduces receptor coupling to G‐proteins. In rat brain, dense A_2A‐AR‐like immunoreactivity that is eliminated by the blocking peptide was found in the neuropil of the striatum, nucleus accumbens (rostral pole, core and shell), cell bridges of the striatum, olfactory tubercles, and areas of extended amygdala with somewhat lighter labeling in the globus pallidus and nucleus of the solitary tract. Light perikaryal labeling was found in other areas of the brain, including the cortex, hippocampus, thalamus, cerebellum, and portions of the hindbrain. The observed distribution of A_2A‐AR immunoreactivity throughout the neuraxis is consistent with the receptors' role in modulating dopaminergic neurotransmission and central control of cardiovascular function. J. Comp. Neurol. 401:163–186, 1998. © 1998 Wiley‐Liss, Inc.