A general approach for reversible affinity labeling of receptors has been developed. The objective is to carry out a series of chemical modifications resulting in a covalently-modified, yet functionally-regenerated, receptor protein that also may contain a reporter group. The ligand recognition site of A1-adenosine receptors in bovine brain membranes was probed to demonstrated the feasibility of this approach. Use of disulfide or ester linkages, intended for cleavage by exposure of the labeled receptor to either reducing reagents or hydroxylamine, respectively, was considered. Binding of the antagonist radioligand [3H]CPX was preserved following incubation of the native receptor with 3 M hydroxylamine, while binding was inhibited by the reducing reagent dithiothreitol (DTT) with an IC50 of 0.29 M. Hydroxylamine displaced specific agonist ([3H]PIA) binding in a noncovalent manner. Specific affinity labels containing reactive isothiocyanate groups were synthesized from XCC (8-[4-](carboxymethyl)-oxy]phenyl]-1,3-dipropylxanthine) and shown to bind irreversibly to A1-receptors. The ligands were structurally similar to previously reported xanthine inhibitors (e.g., DITC-XAC: (1989) J. Med. Chem. 32, 1043) except that either a disulfide linkage or an ester linkage was incorporated in the chain between the pharmacophore and the isothiocyanate-substituted ring. These groups were intended for chemical cleavage by thiols or hydroxylamine, respectively. Radioligand binding to A1-receptors was inhibited by these reactive xanthines in a manner that was not reversed by repeated washing. Hydroxylamine or DTT restored a significant fraction of the binding of [3H]CPX in A1-receptors inhibited by the appropriate cleavable xanthine isothiocyanate derivative.