Many synaptic connections are rejected during development, and the remainder are stabilized. Whether neuronal activity is important in this remodeling remains unknown. Cholinergic synapses are formed in tissue culture between the hybrid NG108–15 (neuroblastoma X glioma) cell and skeletal myotubes. We have investigated changes in these synapses brought about by chronic depolarization. Most myotubes are innervated under control conditions and recording from a myotube while stimulating a neighboring hybrid cell demonstrates that most hybrid- myotube pairs in anatomical proximity also are connected synaptically. Multiple innervation of one myotube by several hybrid cells is common. After 24 to 72 hr of cell depolarization with low concentrations of veratridine, myotubes continue to evidence synaptic activity, but the chance of evoking activity, in a given myotube by stimulation of a neighboring hybrid cell, is diminished to 30% of control values; only 5% of myotubes can be demonstrated to still have multiple innervation. However, the efficacy of synapses that persists after veratridine exposure is comparable to control. By 24 hr after removal of veratridine, synapse number returns to control levels. Tetrodotoxin prevents these effects. We suggest that components responsible for the well known development change from polyneuronal to mononeuronal innervation may be present and accessible to manipulation in this relatively well defined tissue culture system.