Incubation of minced mouse-forebrain tissues in lithium Krebs solution reduces the acetylcholine content of the vesicular fraction 70 percent without altering that of the cytoplasmic fraction. Depleted vesicular-bound acetylcholine can be restored with newly synthesized acetylcholine (formed from extracellular choline) independently of the cytoplasmic pool. Depletion of vesicular-bound acetylcholine does not facilitate the movement of preformed extracellular acetylcholine into vesicles.