AbstractModulation by exogenous and endogenous adenine nucleotides and adenosine of [3H]acetylcholine release evoked by veratridine (10 μM) was compared in synaptosomal fractions from the hippocampus and the cerebral cortex of the rat. In both brain areas, exogenously added ATP or adenosine (10–100 μM) inhibited the evoked tritium release. In the hippocampus, ATPμS, an ATP analogue more resistant to catabolism than ATP, was virtually devoid of effect on tritium release, and the effect of ATP was prevented by the ecto‐5′‐nucleotidase inhibitor α,β‐methylene ADP (100 μM), by adenosine deaminase (2 U/ml) and by the A1adenosine receptor antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX, 20 nM). In contrast, in the cerebral cortex, the effect of ATP on tritium release was not prevented by either α,β‐methylene ADP (100 μM) or adenosine deaminase (2 U/ml), and several ATP analogues (30 μM) inhibited release. The order of intensity of the inhibitory effects of the ATP analogues was: ATPγS>ATP>ñ,γ‐imido ATP>β,γ‐methylene ATP>2‐methyl‐S‐ATP, α,β‐methylene ATP. The effect of ATPγS in the cerebral cortex was prevented by DPCPX (20 nM) and was not affected by the P2purinoceptor antagonist suramin (100 μM). In the hippocampus, α,β‐methylene ADP (100 μM) increased the evoked release of tritium, and adenosine deaminase (2 U/ml) produced an even greater increase; when adenosine deaminase was added in the presence of α,β‐methylene ADP, adenosine deaminase still increased the evoked release of tritium. In the cerebral cortex, DPCPX (20 nM) and adenosine deaminase (2 U/ml) increased the evoked tritium release by a similar magnitude, but the effect of adenosine deaminase was smaller than in the hippocampus. It is concluded that in the cerebral cortex ATP as such presynaptically inhibits acetylcholine release, whereas in the hippocampus the role of adenine nucleotides is as a source of endogenous extracellular adenosine that tonically inhibits acetylcholine release. The results also show that besides formation of adenosine from adenine nucleotides, released adenosine as such contributes in nearly equal amounts to the pool of endogenous adenosine that presynap