Abstract A [ 3H]xanthine amine congener (XAC), a potent adenosine receptor antagonist, binds in a saturable and reversible fashion to high affinity binding sites in mouse brain ( B max = 323 ± 17 fmol/mg protein, K d = 1.4 ± 0.4 nM). Adenosine receptor agonists and antagonists are more potent than adenosine uptake inhibitors in displacing the binding of [ 3H]xanthine amine congener ([ 3H]XAC). The anatomical distribution of [ 3H]XAC binding sites is consistent with its being a ligand probe for adenosine receptors. High binding site densities were observed in the hippocampus (stratum oriens and radiatum, molecular layer), superior colliculus (superficial gray), cerebellum (molecular layer), cerebral cortex and substantia nigra. The availability of a high affinity antagonist radioligand probe like [ 3H]XAC for adenosine receptors allows the comparative quantitative autoradiographic analysis of agonist and antagonist binding to adenosine receptors, e.g. under varying in vitro incubation conditions (presence and absence of guanine nucleotides and cations).