Adenosine modulates neuronal activity and neurotransmitter release through interaction with cell surface receptors. Four adenosine receptor subtypes, A1, A2A, A2B, and A3 receptors, have been cloned and characterized. The agonist ligand, [125I]AB-MECA ([125I]4-aminobenzyl-5'N-methylcarboxamidoadenosine) has high affinity for recombinant A1 and A3 receptors [Olah et al., Mol. Pharmacol, 45 (1994) 978-982]. Rodent A3 receptors are relatively insensitive to xanthines; inhibition of A1 receptors with xanthines allows selective detection of A3 receptors despite the lack of selectivity of the ligand. We studied whether [125I]AB-MECA is useful for localization and characterization of A3 receptors in rat brain. The autoradiographic distribution of total [125I]AB-MECA (400 pM) binding closely resembled the pattern of A1 receptor binding, with highest levels in cerebellum, hippocampus, and thalamus, and moderate levels in cortex and striatum. Drug competition studies confirmed that almost all [125I]AB-MECA binding could be attributed to labeling of A1 receptors. Xanthine amine congener (1 microM) reduced specific [125I]AB-MECA binding by > 95%, indicating that xanthine-resistant A3 receptors represent a quantitatively minor subtype. Despite the use of a radioligand with high affinity and high specific activity, the low density of A3 receptors in rat brain appears insufficient to allow localization, or even consistent detection, of this receptor subtype. In the presence of DPCPX (50 nM, to block A1 receptors), residual [125I]AB-MECA binding to A2A receptors was observed in the striatum. Thus [125I]AB-MECA labels primarily A1 and A2A adenosine receptors in rat brain.