A newly synthesized, chemically reactive adenosine derivative, N6-(3-isothiocyanatobenzyl)adenosine-5'-N-methyluronamide, was found to bind selectively to A3 receptors. Ki values for this isothiocyanate derivative in competition binding at rat brain A1, A2a, and A3 receptors were 145, 272 and 10.0 nM, respectively. A preincubation with this derivative resulted in irreversible inhibition of radioligand binding at rat A3 receptors in membranes of transfected CHO cells or RBL-2H3 mast cells, but not at rat A1 or A2a receptors. The loss of binding sites for 0.1 nM [125I]N6-(4-aminobenzyl)adenosine-5'-N-methyluronamide, a high affinity A3 receptor radioligand, in transfected CHO cell membranes was concentration-dependent with an IC50 of 50 nM. No change was observed in the Kd value of the remaining A3 receptor sites. The inhibition was also insensitive to theophylline (1 mM), consistent with the pharmacology of rat A3 receptors. Structurally similar adenosine analogues lacking the chemically reactive isothiocyanate group failed to irreversibly inhibit A3-binding.