Two isomeric isothiocyanate derivatives of the A1 adenosine receptor antagonist xanthine amine cogener (XAC) have been synthesized and found to be potent affinity labels (irreversibly bound ligands) for A1 adenosine receptors. The interaction of m- and p-isomers of 1,3-dipropyl-8-isothiocyanatophenyl(aminothiocarbonyl (2-aminoethylaminocarbonyl(4-methyloxy(phenyl]])-xanthine (DITC-XAC) with rat brain A1 receptors is of high affinity (EC50 = 27 and 52 nM, respectively) as determined by radioligand competition curves. These compounds reduced the number of A1 receptors (greater than 90% at 500 nM m-DITC-XAC) in brain membranes, without any change in the affinity of the remaining receptors for [125I]N6-2-(4-aminophenyl)ethyladenosine. Prior reaction of the isothiocyanate moiety with ethylenediamine did not alter the affinity of the XAC derivative for the A1 receptor but eliminated its ability to covalently incorporate into the receptor. Incubation of brain membranes with radiolabeled p- and m-DITC-XAC results in the specific labeling of a Mr 38,000 peptide. This labeling can be blocked with both an A1 adenosine receptor-specific agonist and an antagonist. This specific protein has the same molecular weight as the protein labeled with A1-selective photoaffinity probes. The much higher efficiency of incorporation of these affinity probes compared with photoaffinity probes should make them extremely useful for structural studies of A1 adenosine receptors.