A series of highly specific inhibitors and substrates were used to specifically characterize the esterases present in the five vestibular end organs (the three semicircular canal cristae, the utriclar, and the saccular macula) from four young adult cats. No butyrylcholinesterase or nonspecific acetylcholinesterase activity was found. The results indicate a dense localization of acetylcholinesterase activity beneath and between the hair cells, at the sensory epithelial and subepithelial junction, and within vascular elements. This reaction product extends along the entire length of the sensory epithelium. Activity also extends proximally to the areas of the nerves' entrance into the subepithelial tissue. It should be noted that most of the activity is likely to be located in the extracellular matrix, as well as in the endoneurium. This extracellular distribution may represent an enzymatic barrier to the diffusion of acetylcholine that may be essential for the normal function of the end organ and possibly similar to the blood-brain barrier in some respects.