The role of innervation in the establishment and regulation of the synaptic density of voltage-activated Na channels (NaChs) was investigated at regenerating neuromuscular junctions. Rat muscles were induced to degenerate after injection of the Australian tiger snake toxin, notexin. The loose-patch voltage clamp technique was used to measure the density and distribution of NaChs on muscle fibers regenerating with or without innervation. In either case, new myofibers formed within the original basal lamina sheaths, and, NaChs became concentrated at regenerating endplates nearly as soon as they formed. The subsequent increase in synaptic NaCh density followed a time course similar to postnatal muscles. Neuromuscular endplates regenerating after denervation, with no nerve terminals present, had NaCh densities not significantly different from endplates regenerating in the presence of nerve terminals. The results show that the nerve terminal is not required for the development of an enriched NaCh density at regenerating neuromuscular synapses and implicate Schwann cells or basal lamina as the origin of the signal for NaCh aggregation. In contrast, the change in expression from the immature to the mature form of the NaCh isoform that normally accompanies development occurred only partially on muscles regenerating in the absence of innervation. This aspect of NaCh regulation is thus dependent upon innervation.