The adenosine receptor agonist 2-[ p-(2-carboxyethyl)phenylethylamino]-5'- N-ethylcarboxamidoadenosine (CGS 21680) is generally considered to be a selective adenosine A(2A) receptor ligand. However, the compound has previously been shown to exhibit binding characteristics that are not compatible with adenosine A(2A) receptor binding, at least in brain regions other than the striatum. We have examined binding of [(3)H]CGS 21680 and of antagonist radioligands with high selectivity for adenosine A(1) or A(2A) receptors to hippocampus and striatum of mice lacking either adenosine A(1) (A1R((-/-))) or A(2A) (A2AR((-/-))) receptors. Both receptor autoradiography and membrane binding techniques were used for this purpose and gave similar results. There were no significant changes in the binding of the A(1) receptor antagonist [(3)H]DPCPX in mice lacking A(2A) receptors, or in the binding of the A(2A) receptor antagonists [(3)H]SCH 58261 and [(3)H]ZM 241385 in mice lacking A(1) receptors. Furthermore, [(3)H]CGS 21680 binding in striatum was abolished in the A2AR((-/-)), and essentially unaffected in striatum from mice lacking A(1) receptors. In hippocampus, however, binding of [(3)H]CGS 21680 remained in the A2AR((-/-)), whereas binding was virtually abolished in the A1R((-/-)). There were no adaptive alterations in A(2A) receptor expression in this region in A1R((-/-)) mice. Thus, most of the [(3)H]CGS 21680 binding in hippocampus is dependent on the presence of adenosine A(1) receptors, but not on A(2A) receptors, indicating a novel binding site or novel binding mode.