During neuromuscular synapse development, the degradation rate of ACh receptors (AChRs) accumulated in the synaptic portion of the muscle membrane is drastically reduced under neural control, their half-life t1/2 increasing from 1 d to about 12 d. Recent evidence suggests that the metabolic stability of synaptic AChRs is mediated by the muscle activity induced by the nerve. We have now investigated the pathway linking muscle activity and metabolic stabilization of synaptic AChRs in organ cultured rat muscle. Soleus and diaphragm muscles were denervated for 14-40 d, a procedure leading to the destabilization of synaptic AChRs, and conditions required to restabilize synaptic AChRs in the denervated muscle were analyzed. The activity-dependent stabilization of synaptic AChRs in chronically denervated endplates required calcium entry through dihydropyridine-sensitive Ca2+ channels activated by high-frequency stimulation for approximately 6 hr and was specific for synaptic AChRs. As in vivo, extrasynaptic AChRs were not stabilized, and their t1/2 remained 1 d. The stabilization process was not dependent on de novo protein synthesis, and it could also be brought about by elevated cAMP levels. Furthermore, it required shorter stimulation periods in the presence of the phosphatase inhibitors okadaic acid and calyculin A, whereas blockade of protein kinases with high doses of staurosporine blocked the stabilization. Activity- dependent, dihydropyridine-sensitive as well as cAMP-dependent phosphorylation of myosin light chain was observed. These findings are consistent with the notion that muscle activity initiates AChR stabilization via the activation of calcium-dependent protein phosphorylation reactions.