Brief and chronic exposure of rats to neostigmine methylsulfate produced marked morphological alterations of the fine structure at the end-plate region of the extensor digitorum longus muscles. These changes were dose and time dependent and were restricted primarily to the subjunctional myofibrillar apparatus and membrane-bound organelles. In addition, significant presynaptic alterations were observed including synaptic vesicle depletion and the appearance of numerous coated vesicles and membrane cisternae, which indicated continuing nerve terminal hyperactivity. With chronic treatment, degeneration and partial recovery of the nerve axon also were observed. The morphological changes of the end-plate region induced by neostigmine did not occur in most fibers after brief denervation and were eliminated entirely by chronic nerve section. Thus, the postsynaptic degenerative changes caused by neostigmine treatment observed in nondenervated animals appear to result primarily from greatly increased synaptic activity and not primarily from a direct neostigmine reaction with the pre- or postsynaptic membranes. Since the myopathic changes observed in this study were produced by neostigmine, a drug which is commonly employed in the routine treatment of human patients with myasthenia gravis, continued use of neostigmine for long-term therapy in noncrisis situations may not be accepted as being free from risk.