Muscle fibers in the soleus muscle of the rat, injured by bupivacaine and free autografting, were allowed to regenerate within their old basal laminae. Histochemical and cytochemical analysis of newly synthesized acetylcholinesterase (AChE) revealed that two kinds of focal accumulations of AChE appeared in regenerating myotubes. First, AChE gets concentrated at the sites of the former motor endplates. Accumulation of AChE starts in places where a tight contact between the remnants of the old junctional basal lamina and the budding surface of the myotube engulf the extracellular material. Appearance of these AChE accumulations can be prevented by papain treatment of the soleus muscle before autografting but not by predenervating it for 1 month. Focalization of AChE is probably induced by a component of the junctional basal lamina, possibly a protein, the existence of which is not dependent upon continuous presence of the motor nerve and may be produced by the muscle. This view is corroborated by the fact that an additional kind of AChE accumulation appeared in regenerating muscles in regions remote from the sites where motor endplates were located in the muscles of origin. Although differing in localization, size, and appearance, both kinds of AChE accumulations ultrastructurally resemble the postsynaptic specialization of the motor endplate: they consist of tubelike sarcolemmal invaginations containing AChE. The extrajunctional AChE accumulations seem to arise spontaneously and are usually located more than 750 micron away from the junctional ones as if some local inhibitory mechanism prevents their formation in the immediate vicinity.