The effects of choline (Ch) on the spontaneous release of endogenous gamma-aminobutyric acid (GABA) and of 3H-GABA were studied in superfused rat hippocampal synaptosomes. Choline enhanced in a concentration-dependent way the release of endogenous GABA but did not affect that of the radioactive aminoacid. The effect of Ch was not antagonized by atropine or mecamylamine; moreover, it was not mimicked by acetylcholine, oxotremorine or carbachol. The Ch-induced GABA release was counteracted by hemicholinium-3. Thus the release of endogenously synthesized GABA (but not that of the aminoacid taken up) may be regulated by Ch through a mechanism involving penetration into the releasing terminal through a Ch uptake system.