[3H]NECA (1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuronamide) is known to bind to both the A1 and A2 subtypes of adenosine receptor in rat striatal membranes. In order to study the putative A2 component of [3H]NECA binding, we examined several compounds for the ability to selectively eliminate the A1 component of binding; N6-cyclopentyladenosine was found to give the most satisfactory results. Binding of [3H]NECA in the presence of 50 nM N6-cyclopentyladenosine was characterized. The rank order of potency for inhibition of [3H]NECA binding was NECA much greater than 2-chloroadenosine greater than N6-[(R)-1-methyl-2-phenyl-ethyl]adenosine (R-PIA) greater than N6-cyclohexyladenosine greater than S-PIA, indicating that binding was to an A2 adenosine receptor. When affinities of compounds in [3H]NECA binding to A2 receptors were compared to their affinities in [3H]N6-cyclohexyladenosine binding to A1 receptors, N6-cyclopentyladenosine was the most A1-sensitive agonist (A1 Ki, 0.59 nM; A2 Ki, 460 nM; Ki ratio, 780), whereas the selective coronary vasodilator 2-(phenylamino)adenosine was the most A2-selective agonist (A1, 560 nM; A2, 120 nM; ratio, 0.21). The antagonist 8-cyclopentyltheophylline had considerable A1 selectivity (A1, 11 nM; A2, 1400 nM; ratio, 130), whereas alloxazine had slight A2 selectivity (A1, 5200 nM; A2, 2700; ratio, 0.52). [3H]NECA binding to A2 receptors was highest in striatum but was detectable at much lower levels in each of seven other brain areas. The regional distribution of [3H]NECA binding and the affinities of adenosine agonists and antagonists for inhibition of binding indicate that the site labeled by [3H]NECA belongs to the high affinity, or A2a, subclass of A2 receptor.