This protocol describes reconstitution assays to study how the neurotransmitter release machinery triggers Ca2+-dependent synaptic vesicle fusion. The assays monitor fusion between proteoliposomes containing the synaptic vesicle SNARE synaptobrevin (with or without the Ca2+ sensor synaptotagmin-1) and proteoliposomes initially containing the plasma membrane SNAREs syntaxin-1 and soluble NSF attachment protein (SNAP)-25. Lipid mixing (from fluorescence de-quenching of Marina-Blue-labeled lipids) and content mixing (from development of fluorescence resonance energy transfer (FRET) between phycoerythrin-biotin (PhycoE-Biotin) and Cy5-streptavidin trapped in the two proteoliposome populations) are measured simultaneously to ensure that true, nonleaky membrane fusion is monitored. This protocol is based on a method developed to study yeast vacuolar fusion. In contrast to other protocols used to study the release machinery, this assay incorporates N-ethylmaleimide sensitive factor (NSF) and α-SNAP, which disassemble syntaxin-1 and SNAP-25 heterodimers. As a result, fusion requires Munc18-1, which binds to the released syntaxin-1, and Munc13-1, which, together with Munc18-1, orchestrates SNARE complex assembly. The protocol can be readily adapted to investigation of other types of intracellular membrane fusion by using appropriate alternative proteins. Total time required for one round of the assay is 4 d.