Although evidence suggests that insulin-like growth factor plays an important role in the development and growth of the nervous system, the effect of IGF-1 in the regulation of neurotransmitter release in the peripheral nervous system remains unknown. Here we show that acute application of insulin-like growth factor-1 (IGF-1), a factor widely expressed in developing myocytes, dose-dependently enhances the spontaneous acetylcholine (ACh) secretion at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch clamp recording. We studied the role of endogenously released IGF-1 by examining the effect of IGF-1 antibody on the frequency of spontaneous synaptic currents (SSCs) at high-activity synapses, and found SSC frequency was markedly reduced at these high-activity synapses. The IGF-1-induced synaptic potentiation was not abolished when calcium was eliminated from the culture medium or there was bath application of the pharmacological calcium channel inhibitor cadmium, indicating that calcium influxes through voltage-activated calcium channels are not required. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP₃) or ryanodine receptors effectively occluded the increase of SSC frequency elicited by IGF-I. Treating cells with the phosphoinositide-3 kinase (PI3-K) inhibitors wortmannin or LY294002, and with phospholipase C-gamma (PLC-gamma) inhibitor U73122, but not inhibitor of mitogens-activated protein (MAP) kinase PD98059 abolished IGF-1-induced synaptic potentiation. Taken collectively, these results suggest that endogenously released IGF-1 from myocytes elicits calcium release from IP₃ and/or ryanodine sensitive intracellular calcium stores of the presynaptic nerve terminal. This is done via PI3 kinase and PLC-gamma signaling cascades, leading to an enhancement of spontaneous transmitter release.