Membrane fusion is an essential biological process for vesicle trafficking in eukaryotic cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of proteins that have been recognized as key components of protein complexes that drive membrane fusion. The SNARE proteins localized in opposite membranes form a SNARE complex, which bridges the two membranes into close proximity and promotes fusion. Despite considerable sequence divergence among SNARE proteins, this fusion machinery is conserved structurally, mechanistically and evolutionally from yeast to human;Site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) are well established techniques to study the structures of membrane proteins. Fluorescent labeled lipids allow us to test the function of SNARE proteins by lipid mixing assay. With these techniques, we are able to study the structures and functions of SNAREs in vitro. In this dissertation, three sets of SNARE proteins coming from yeast, plant and mammalian sources have been investigated. The information gathered from these systems will help to understand the molecular mechanism of SNARE-induced membrane fusion.