The purpose of this course of study is to understand the intermediates during SNAREs assembly and the membrane fusion mediated by SNAREs. SNAREs have been proposed to be the minimal machinery for the membrane fusion. According to the location distribution, SNAREs can be classified as either v-(vesicles) or t-(target) SNAREs. The assembly of three SNARE components from the opposing membranes into the ternary complex is believed to provide the ultimate driving force for merging the separate bilayers into a continuous entity. Before this step the t-SNAREs associate as the binary complex on the target membrane to serve as the intermediate for the oncoming v-SNARE. To better understand the assembly process of the neuronal SNAREs, the structure of binary complex was analyzed by EPR method. In general, the binary complex was revealed to share similar four-stranded helix bundle structure with the ternary complex;The lipid mixing during the membrane fusion was observed by two methods: the bulk assay and the single fusion assay. The mutant yeast v-SNARE with truncated transmembrane domain or introduction of special lipid plus low SNAREs surface density led to the discovery of hemifusion intermediate in the fusion pathway by the bulk assay. The results from the bulk assay for the neuronal SNAREs also contended with the hemifusion model. In order to elucidate the lipid mixing during the fusion on the molecular level, a new generation single fusion assay was developed by monitoring lipid mixing in real-time FRET on the single liposome level. The results affirmed that hemifusion exists as the on-pathway intermediate and post-hemifusion intermediates were characterized.