Although molecular biology studies indicate the presence of adenosine A(2A) receptors in the rat hippocampus, the pharmacological characterization of adenosine A(2A) receptor binding and of its putative facilitatory effects has revealed features essentially different from these found for adenosine A(2A) receptors in most preparations. We now confirmed that activation of adenosine A(2A) receptors with 2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, 1-30 nM) or 2-hexynyl-5'-N-ethylcarboxamidoadenosine (HENECA, 3-100 nM) facilitated the veratridine-evoked [3H]acetylcholine release from hippocampal synaptosomes with a maximal effect of 14+/-2% and 16+/-2%, respectively. These effects were prevented by the adenosine A(2A) receptor antagonists, 4-(2-[7-amino-2-[2-furyl][1,2,4]-triazolo[2,3a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM 241385, 20 nM) and 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261, 20 nM), but not by the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 20 nM). Adenosine A(2A) receptors may activate adenylate cyclase and protein kinase A since CGS 21680 (10 nM) facilitation of [3H]acetylcholine release was occluded by 8-bromo-cAMP (0.5 mM) and forskolin (10 microM) and prevented by H-89 (1 microM), but unaffected by phorbol-12,13-didecanoate (250 nM) or bisindolylmaleimide I (1 microM). The existence of adenosine A(2A) receptors in hippocampal nerve terminals was further confirmed by a Western blot immunoreactivity qualitatively identical to that found in the striatum. This constitutes the first pharmacological identification of canonical adenosine A(2A) receptors coupling to the expected cAMP/protein kinase A pathway in the hippocampus with the expected immunoreactivity.