After collagenase treatment and mechanical disruption, acetylcholinesterase (AChE) activity on the surface of individual flexor brevis muscle fibers fell by 88%. During the next 48 hr in culture, surface AChE activity continued to decline, while intracellular activity changed little. After 1 week in culture total muscle fiber AChE activity fell to very low levels and intracellular AChE activity could no longer be detected, probably as a result of reduced synthesis and rapid externalization of intracellular AChE. Apart from the removal of most of the surface activity, cultured muscle fibers had similar AChE activity to muscle fibers that had been denervated in vivo, suggesting that the changes observed in culture reflect the loss of neuromuscular interaction and not to any contributory effects of the dissociation process. It is to be hoped that these results, along with the published results of Bekoff and Betz [J. Physiol, 271:25-40, 537-547], will serve as useful background data for those continuing to use adult dissociated muscle fibers in their studies.