The rat A 3 adenosine receptor (AR) is a recently characterized AR subtype cloned from testis and brain cDNA libraries. N 6-2-(4-Amino-3-[ 125I]iodophenyl)ethyladenosine, a high affinity A 1AR agonist, has served as the only radioligand available for study of the A 3AR. The relatively low affinity of N 6-2-(4-amino-3-[ 125I]iodophenyl)ethyladenosine for the A 3AR and its greater A 1AR selectivity necessitate the development of more appropriate radioligands for A 3AR analysis. This report characterizes 125I-4-aminobenzyl-5′- N-methylcarboxamidoadenosine ( 125I-AB-MECA), a high affinity radioligand for the A 3AR, in two cell lines that express this AR subtype. Membranes from Chinese hamster ovary (CHO) cells expressing the rat A 3AR and from the rat mast cell line RBL-2H3 bound 125I-AB-MECA with K d values of 1.48 ± 0.33 nM and 3.61 ± 0.30 nM, respectively. As determined by 125I-AB-MECA binding, levels of A 3AR expresssion in the A 3AR-CHO cell line and RBL-2H3 cells were 3.06 ± 0.21 pmol/mg and 1.02 ± 0.13 pmol/mg, respectively. Binding of 125I-AB-MECA was characterized in competition assays. In the A 3AR-CHO cell line a potency order of cyclohexyl-5′- N-ethylcarboxamidoadenosine (cyclohexyl-NECA) = benzyl-NECA > (−)- N 6-[( R)-phenylisopropyl]adenosine = NECA was observed, and in RBL-2H3 cells (−)- N 6-[( R)-phenylisopropyl]adenosine and NECA were equipotent. Xanthine amine congener (XAC) and 8-cyclopentyl-1,3-dipropylxanthine did not significantly inhibit 125I-AB-MECA binding. The parent compound, AB-MECA, dose-dependently inhibited forskolin-stimulated adenylyl cyclase activity in A 3AR-CHO cell membranes. 125I-AB-MECA bound to the rat A 1AR and canine A 2aAR expressed in COS-7 cells with K d values of 3.42 ± 0.43 nM and 25.1 ± 12.6 nM, respectively. This binding was significantly reduced in the presence of 1 μM XAC. In RBL-2H3 cells, XAC had no effect on 125I-AB-MECA affinity and reduced the level of radioligand binding by ~5%.