Calcium ions are important in many vital neuron processes, including spontaneous neurotransmit-ter release. Extracellular calcium has long been known to be related to spontaneous neurotransmitter re-lease, but the detailed mechanism for the effect of intracellular Ca2+ on synaptic release has not yet been understood. In this research, 1,2-bis-(o-aminophenoxy)-ethane-N, N, N’, N’-tetraacetic acid tetraacetoxy-methyl ester (BAPTA-AM) was used to combine with cytosolic free Ca2+ in a calcium free medium of cul-tured Xenopus neuromuscular junctions (NMJ). The spontaneous synaptic current (SSC) frequency was ob-viously reduced. Then, drugs were applied to interrupt and activate the Ca2+ release channels in the endo-plasmic reticulum (ER) membrane, but the SSC frequency was not affected. The results show that sponta-neous neurotransmitter release depends on intracellular rather than ER calcium in cultured Xenopus NMJ without extracellular calcium.