The A2a adenosine receptor (AR) mediates several important physiological effects of adenosine, including vasodilation and inhibition of platelet aggregation. Until recently, no antagonist radioligand of sufficient selectivity or affinity was available. We describe the synthesis and characterization by radioligand binding of 125I-4-(2-[7-amino-2-{2-furyl}-{1,2,4}triazolo{2,3-a}- {1,3,5}triazin-5-yl-amino]ethyl)phenol (125I-ZM241385) in membranes from two cell types that express A2a ARs. Membranes from Chinese hamster ovary (CHO) cells expressing a recombinant canine A2a AR bound 125I-ZM241385 with high affinity, and agonist competition experiments with 2-(p-carboxyethyl)-phenylamino-5'-N-carboxamidoadenosine, 5'-N-ethylcarboxamidoadenosine, and (-)-N6[(R)-phenylisopropyl]adenosine revealed a potency order characteristic of an A2a AR binding site. Membranes from bovine striatum, which contain a native A2a AR, also bound 125I-ZM214385 with similarly high affinity and also displayed a pharmacological profile for displacement of radioligand binding that was consistent with that of an A2a AR. Also, under conditions in which 125I-ZM241385 bound with high affinity to a recombinant rat A2a AR expressed in CHO cells, no specific binding was detectable in membranes from CHO cells expressing functional rat A1, A2b, or A3 ARs, indicating that over the range of concentrations used in radioligand binding assays, 125I-ZM214385 is a highly selective antagonist radioligand for study of A2a ARs within a given species.