Abstract Acetylcholinesterase (AChE) inhibitors which differ in lipid solubility and thus in their ability to penetrate cell membranes were utilized to study the effects of denervation on discrete pools of 16S AChE from endplate regions of adult rat anterior gracilis muscle. Such pools have been interpreted as extra- and intracellular fractions of endplate 16S AChE activity. Denervation caused an almost immediate decay of intracellular 16S AChE, and a later (12–18 h) but roughly parallel decrease in its extracellular counterpart. Thus, the level at which the motor nerve exerts its primary regulatory influence on adult mammalian skeletal muscle endplate 16S AChE activity appears to be within muscle cells. This influence may affect the molecule's synthesis, assembly, and/or degradation.