At the synapse, neurotransmitters are released via Ca2+-triggered exocytotic fusion of synaptic vesicles with the presynaptic plasma membrane. The whole process is controlled by various proteins. SNAREs have been recognized as the key components that drive membrane fusion. In addition, many other proteins, such as Munc18/nSec1, Mun13, synaptotagmin, complexin, etc. are characterized to regulate synaptic transmission temporally and spatially.;The in vitro bulk fluorescence assay was applied to examine the kinetics of membrane fusion of liposomes mediated by recombinant neuronal SNAREs and led to the demonstration of hemifusion as an intermediate in the pathway. In order to monitor the fusion process in more sophisticated level, we developed the Single-liposome FRET assay and combined site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) techniques to study the function of two SNARE regulators, synaptotagmin and complexin.;The EPR and fluorescence assay were also applied for the study of the SNAREs mediating the trafficking in yeast. It was found that supermolecular SNARE assembly precedes hemifusion, which was subsequently followed by distal leaflet mixing and formation of the cis-SNARE complex.