Reconstitution assays with proteoliposomes provide a powerful tool to elucidate the mechanism of neurotransmitter release, but it is important to understand how these assays report on membrane fusion, and recent studies with yeast vacuolar SNAREs uncovered asymmetry in the results of lipid mixing assays. We have investigated whether such asymmetry also occurs in reconstitution assays with the neuronal SNAREs, using syntaxin-1-SNAP-25-containing liposomes and liposomes containing synaptobrevin (T and V liposomes, respectively), and fluorescent probes to monitor lipid and content mixing simultaneously. Switching the fluorescent probes placed on the T and V liposomes, we observed a striking asymmetry in both lipid and content mixing stimulated by a fragment spanning the two C2 domains of synaptotagmin-1, or by a peptide that spans the C-terminal half of the synaptobrevin SNARE motif. However, no such asymmetry was observed in assays performed in the presence of Munc18-1, Munc13-1, NSF and αSNAP, which coordinate the assembly-disassembly cycle of neuronal SNARE complexes. Our results show that switching fluorescent probes between the two types of liposomes provides a useful approach to better understand the reactions that occur between liposomes and detect heterogenous behavior in these reactions.