The investigations were carried out on white rats determining the level and synthesis of acetylcholine (ACh) in the cerebral cortex, striatum, and in some experiments also in the brain stem. Thyroxine administered subcutaneously increased ACh synthesis in the cerbral cortex and reduced it in the striatum without changing the level of ACh in these structures. After thyroxine administration in vitro these changes were not observed. Intraperitoneal insulin caused no changes in the level and synthesis of ACh while in vitro ACh synthesis was increased in the cortex as well as striatum after insulin. Glucagon, hydrocortisone, adiuretin and oxytocin had no effect on ACh level and synthesis in the tested structures.