The intrinsic affinity of 8-phenylxanthine analogs at striatal A(2)-adenosine receptors is highly species dependent. [(3)H]XAC (8-[2-aminoethyl[amino[carbonyl[methyl[oxyphenyl]]]]]-1,3-dipropylxanthine), although A(1)-selective in the rat brain, binds to A(2) receptors in rabbit striatal membranes with sufficiently high affinity to serve as a radioligand. In the presence of 50 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), an A(1)-selective antagonist added to eliminate binding to A(1) receptors, [(3)H]XAC exhibits saturable, specific binding (70% of total) to A(2) sites with a K(d) of 3.8 nM and a B(max) of 1.23 pmol/mg protein. At 24 degrees C, the association and dissociation rate constants were 0.13 min(?1) nM(?1) and 0.36 min(?1), respectively. Binding was performed for 1 h, with non-specific binding defined in the presence of 100 ?M NECA (N-ethylcarboxamidoadenosine). The potency order for antagonists against 1 nM [(3)H]XAC at rabbit A(2)-receptors was XAC ? N(?)-Me-XAC ? CPX = XCC > 1,3-dipropyl-8-p-sulfophenylxanthine > PSPT. The relative potency order for agonists was CGS ? NECA > APEC [= 2-(aminoethylaminocarbonyl-ethylphenylethylamino)-NECA] > PAPA-APEC > ADAC > R-PIA (N(6)-phenylisopropyladenosine) > S-PIA. The ability to characterize central A(2)-adenosine receptors using an antagonist ligand that is chemically functionalized offers the possibility to design affinity labeling probes for this receptor subtype in the brain, similar to those antagonist probes already developed for A(1)-receptors. The results also suggest that affinity columns containing chemically immobilized XAC may be used for isolating central A(2)-adenosine receptors from rabbit striatum.