Neuronal signaling occurs through both action potential-triggered synaptic vesicle fusion and spontaneous release, although the fusion clamp machinery that prevents premature exocytosis of synaptic vesicles in the absence of calcium is unknown. Here we demonstrate that spontaneous release at synapses is regulated by complexin, a SNARE complex-binding protein. Analysis of Drosophila melanogaster complexin null mutants showed a marked increase in spontaneous fusion and a profound overgrowth of synapses, suggesting that complexin functions as the fusion clamp in vivo and may modulate structural remodeling of neuronal connections by controlling the rate of spontaneous release.