Adenosine A1 receptors are known to be widely distributed in various regions of the brain. A2A receptors are enriched in the dopamine-rich areas of the brain, but are also present in rat cortex. Electrically stimulated, perfused rat cortical slices were used to examine the influence of interactions between A1 and A2A receptors on the release of acetylcholine (ACh) from cortical cholinergic nerves. The A1-selective agonist, N6-cyclopentyladenosine (CPA) caused a dose-dependent inhibition of ACh release, which was attenuated in the added presence of the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 1 microM). The inhibitory effects of CPA were unaltered in the added presence of the A2-selective antagonist (E)-8-(3,4-dimethyloxystyryl)-1,3-dipropyl-7-methylxanthine (KF 17837; 1 microM). The A2A-selective agonist 2-[p-(carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), over the concentration range 1 nM to 10 microM, did not significantly alter ACh release when given alone or in the presence of DPCPX or KF 17837. These data suggest that the A(2A) receptors previously identified in rat cortex are not functionally coupled to modulation of ACh release in this tissue. This does not exclude that these receptors may regulate the release of other neurotransmitters.