The nicotinic acetylcholine receptor (AChR) is a substrate for at least three different protein kinases, and phosphorylation of the receptor has been shown to increase its rate of desensitization. However, the first messengers that regulate AChR phosphorylation have not yet been identified. This study demonstrates that calcitonin gene-related peptide (CGRP), a neuropeptide present in the axon terminals of the neuromuscular junction, regulates phosphorylation of the AChR in primary rat myotube cultures. CGRP, in the presence of the phosphodiesterase inhibitor Ro 20-1724, increased phosphorylation of the alpha and delta subunits of the AChR. CGRP-induced phosphorylation of the AChR had the same subunit specificity and temporal sequence as previously observed using forskolin or cAMP analogs. Phosphorylation of the AChR in the presence of CGRP appears to be mediated by CGRP-stimulated increases in cAMP levels leading to activation of cAMP-dependent protein kinase. The present results, taken together with the recent demonstration that CGRP increases the rate of AChR desensitization in mouse myotubes, suggest that CGRP may play a physiological role as a regulator of AChR desensitization by modulating AChR phosphorylation at the neuromuscular junction.