Rolling blackout (RBO) is a putative transmembrane lipase required for phospholipase C-dependent phosphatidylinositol 4,5-bisphosphate–diacylglycerol signaling inDrosophilaneurons. Conditional temperature-sensitive (TS)rbomutants display complete, reversible paralysis within minutes, demonstrating that RBO is acutely required for movement. RBO protein is localized predominantly in presynaptic boutons at neuromuscular junction (NMJ) synapses and throughout central synaptic neuropil, andrboTS mutants display a complete, reversible block of both central and peripheral synaptic transmission within minutes. This phenotype appears limited to adults, because larval NMJs do not manifest the acute blockade. Electron microscopy of adultrboTS mutant boutons reveals an increase in total synaptic vesicle (SV) content, with a concomitant shrinkage of presynaptic bouton size and an accumulation of docked SVs at presynaptic active zones within minutes. Genetic tests reveal a synergistic interaction betweenrboandsyntaxin1ATS mutants, suggesting that RBO is required in the mechanism ofN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated SV exocytosis, or in a parallel pathway necessary for SV fusion. TherboTS mutation does not detectably alter SNARE complex assembly, suggesting a downstream requirement in SV fusion. We conclude that RBO plays an essential role in neurotransmitter release, downstream of SV docking, likely mediating SV fusion.