Vesicular trafficking and exocytosis are directed by the complementary interaction of membrane proteins that together form the SNARE complex. This complex is composed of proteins in the vesicle membrane (v-SNAREs) that intertwine with proteins of the target membrane (t-SNAREs). Here we show that modified synaptic vesicles (mSV), containing v-SNAREs, spontaneously fuse to planar membranes containing the t-SNARE, syntaxin 1A. Fusion was Ca2+-independent and did not occur with vesicles lacking v-SNAREs. Therefore, syntaxin alone forms a functional fusion complex with v-SNAREs. Our functional fusion assay uses synaptic vesicles that are modified, so each fusion event results in an observable transient current. The mSV do not fuse with protein-free membranes. Additionally, artificial vesicles lacking v-SNAREs do not fuse with membranes containing syntaxin. This technique can be adapted to measure fusion in other SNARE systems and should enable the identification of proteins critical to vesicle—membrane fusion. This will further our understanding of exocytosis and may improve targeting and delivery of therapeutic agents packaged in vesicles.