1. Glutamate receptors play important roles in synaptic plasticity and neural development. Here we report that, at the developing neuromuscular synapses in Xenopus cultures, the activation of presynaptic glutamate receptors at motor nerve terminals potentiates spontaneous acetylcholine (ACh) release. 2. Co-cultures of spinal neurons and myotomal muscle cells were prepared from 1-day-old Xenopus embryos. Spontaneous synaptic currents (SSCs) were recorded from innervated myocytes using whole-cell recording. Bath application of glutamate (10 microM) markedly increased the frequency of SSCs, and the action of glutamate was reversible. 3. Pretreatment with 0.3 microM tetrodotoxin, which blocks Na+ channels and the conduction of action potentials, only slightly inhibited the potentiating action of glutamate on SSCs. Furthermore, the enhancement of ACh secretion was much more prominent when glutamate was applied locally to the synaptic region. 4. Three types of glutamate receptor agonists, kainate, quisqualate, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and NMDA (N-methyl-D-aspartate), were effective in inducing the potentiating effect. The ranking order was: glutamate > kainate > NMDA > AMPA > quisqualate. Glycine potentiated the effects induced by NMDA. Metabotropic receptors were not involved in the potentiating action of glutamate. 5. The potentiating effect of glutamate depended on the influx of Ca2+ through both L-type Ca2+ channels and NMDA-gated channels. 6. Since glutamate is known to be co-released with ACh at some cholinergic nerve terminals, the released glutamate may serve as a positive feedback regulation of ACh secretion at developing neuromuscular junctions via its action on presynaptic glutamate receptors.