Neuronal factors co-released with neurotransmitters may play an important role in synapse development and function. Calcitonin gene related peptide (CGRP) and adenosine 5′-triphosphate (ATP), two principal neuromodulators present in the motor nerve terminals, were studied for their roles and mechanisms during early development of neuromuscular synapses in Xenopus nerve–muscle co-cultures. CGRP treatment increased the decay time and amplitude of spontaneous synaptic currents (SSCs) recorded from innervated myocytes, without affecting SSC frequency, suggesting a postsynaptic mechanism. ATP also increased the SSC amplitude and decay time. In addition, ATP was shown to potentiate the responses of isolated myocytes to iontophoretically applied acetylcholine (ACh). Single-channel recording from isolated myocytes showed that both CGRP and ATP specifically increased the open time of embryonic-type, low-conductance ACh channels. Pharmacological experiments suggest that the CGRP actions were mediated by cAMP-dependent protein kinase (PKA), while ATP exerted its effects by binding to P2 purinoceptors and thereby activating protein kinase C (PKC). Moreover, the effects of CGRP and ATP on ACh channel activity were restricted to immature myocytes. Taken together, these results suggest that endogenous CGRP and ATP co-released with ACh from the nerve terminal may promote synaptic development by potentiating postsynaptic ACh channel activity during the early phase of synaptogenesis.Key words: acetylcholine receptor, protein kinase A, protein kinase C, Xenopus, synaptogenesis.