Rabbit retinas were incubated in vitro under conditions known to maintain their physiological function. The acetylcholine stores of the cholinergic amacrine cells were labelled by incubation in the presence of [3H]choline. The tissue was then mounted in a fast-flow superfusion chamber, and the release of [3H]acetylcholine under various conditions was measured by liquid cation exchange or high-voltage electrophoresis. When the retina was stimulated by flashing light, the rate of appearance of radioactive acetylcholine in the superfusate increased, with a latency shorter than the resolution of the system. The rate of release of acetylcholine remained elevated as long as the light was flashing, and returned rapidly to baseline when the light was extinguished. A one minute stimulation with steady light caused a burst of acetylcholine release following stimulus onset and a second, smaller, burst following stimulus cessation. In the presence of 2-amino-4-phosphonobutyrate (APB), an agent known to eliminate selectively the transmission of ON responses to the proximal retina, steady light caused acetylcholine release only at stimulus cessation. Other retinas were labelled with [3H]choline, then incubated for 10-80 min in the presence of flashing light (to promote acetylcholine release) and either control medium or medium containing 100 micron APB (to prevent release from cells activated by stimulus onset). These retinas were quick-frozen, freeze-dried and radioautographed on dry emulsion. In retinas incubated under control conditions [3H]acetylcholine was initially present within two bands within the inner plexiform layer. The two bands became fainter together as the tissue's [3H]acetylcholine was released. APB selectively retarded the depletion of [3H]acetylcholine from the band nearest the ganglion cell layer. We conclude that the displaced cholinergic amacrine cells release acetylcholine at the transient when light appears, and the conventionally placed cholinergic amacrine cells release acetylcholine at the transient when light is extinguished. The retinal ganglion cells that receive a light-driven cholinergic input are distinguished from those that do not by a great sensitivity to slow stimulus motion. It is proposed that the dense plexus of cholinergic dendrites and the transient nature of acetylcholine release combine to create the local subunit that enables detection of motion within regions smaller than those ganglion cells' receptive fields.