The release of [3H]acetylcholine from the chick retina was studied. A 5 mM increase in K+-concentration caused an increased release, which was Ca2+-dependent. The effect of 5 mM K+ was neither potentiated by bicuculline nor inhibited by isoguvacine or muscimol. This indicates that the K+-induced release is not controlled by GABA. However, bicuculline and picrotoxin increased the spontaneous efflux of radioactivity, whereas GABA had no significant effect. The results suggest that cholinergic neurons are tonically inhibited by a continuous release of endogenous GABA. Neither glycine or strychnine, nor dopamine or haloperidol had any effect on the spontaneous release.