The nature of the adenosine receptors involved in the enhancement of acetylcholine release in the hippocampus was studied. The A2A agonist, CGS 21680, increased the veratridine-evoked release of [3H]acetylcholine from hippocampal synaptosomes. This presynaptic effect of CGS 21680 was greater at 3-30 nM than at 100 nM. The excitatory effect of CGS 21680 was antagonised by the A2 antagonist, DMPX (10 microM), and by the A2A antagonist, CSC (200 nM), but not by the A1 antagonist, DPCPX (20 nM). We also found co-expression of A2A and choline acetyltransferase mRNAs in the nucleus of the diagonal band and the medial septum, where the cholinergic cell bodies that project into the hippocampus are located. These results indicate that A2A adenosine receptors are present in cholinergic nerve terminals in the hippocampus and that activation of these receptors enhances acetylcholine release.