The retina is an area of the central nervous system that possesses intrinsic cholinergic neurones which release acetylcholine (ACh) in response to stimulation with flickering light. Using an eye-cup preparation in anaesthetized rabbits we found that when the retina was exposed to the P2-purinoceptor antagonist, PPADS, the light-evoked release of ACh was strikingly increased (by over 40%). In contrast, ATP reduced the light-evoked release of ACh by 20%. The inhibitory effect of ATP was not due to its catabolism to adenosine because it was not affected by the A1-adenosine receptor antagonist, DPCPX, in combination with adenosine deaminase. The actions of both ATP and PPADS were completely blocked by strychnine. We conclude that during physiological stimulation of the retina with light, ATP is co-released with ACh and partially inhibits ACh release by activating (with ACh) an inhibitory glycinergic feedback loop.