The in vitro release of radioactivity by light from rabbit retina was studied after intravitreal injection of [3H]GABA (gamma-aminobutyric acid). The site of uptake of [3H]GABA into the retina after intravitreal injection was checked by autoradiography and was localized at 2 h after the injection mainly in glia but at 4 h in amacrine cells and some ganglion cells. If the retina loaded with [3H]GABA was stimulated by light flashes the efflux of radioactivity increased significantly. Chromatographic analysis of the superfusate demonstrated that [3H]GABA was released by light stimulation. However, the efflux was not elicited by constant light, and was abolished in a medium containing 20 mM Mg++ and 0.2 mM++. When the metabolism of GABA was inhibited by amino-oxyacetic acid, light flashes no longer increased the efflux of radioactivity significantly. No light-evoked release of radioactivity could be demonstrated from glia. Pentobarbitone inhibited the spontaneous efflux and prior anaesthetization with pentobarbitone abolished the light-evoked release. These results support the view that GABA is a retinal neurotransmitter in the rabbit.