There is a longstanding controversy on the identity of synaptic vesicles undergoing spontaneous versus evoked release. A recent study, introducing a new genetic probe, suggested that spontaneous release is driven by a resting pool of synaptic vesicles refractory to stimulation. We found that cross-depletion of spontaneously or actively recycling synaptic vesicle pools occurred on stimulation in rat hippocampal neurons and identified the recycling pool as a major source of spontaneous release.