Abstract: The fusion of synaptic vesicles with the pre-synaptic plasma membrane is mediated by SNARE proteins. This work provides key insights into the behaviour of synaptobrevin, the SNARE protein localized to synaptic vesicles, in its native membrane organelle and when it is reconstituted in liposomes. The reactivity of reconstituted synaptobrevin has remained controversial. Recent studies have suggested that synaptobrevin inserted in membranes does not readily engage in SNARE complexes (Hu et al., 2002; Kweon et al., 2003b). I therefore explored the binding characteristics and assembly pathway of the SNAREs on synaptobrevin-bearing membranes. Like its soluble domain, synaptobrevin anchored in membranes binds to the syntaxin/SNAP-25 acceptor complex to form the stable tetra helical coiled coil SNARE bundle. Kinetic simulations and fitting of experimental data helped unravel the SNARE assembly pathway on membranes. Monitoring the effects of endogenous and extraneous factors on SNARE complex assembly suggested that assembly is a robust process, largely unaffected by brain cytosolic factors, membrane fluidity, chaotropicity, divalent ions and lipid composition but is considerably enhanced in the presence of weak counter-ions. In agreement with these observations, SNARE complex assembly rate on liposomes reconstituted with synaptobrevin and on synaptic vesicles was comparable. Being highly reactive molecules, the regulation of SNAREs has considerable importance. Synaptobrevin has been found to be associated in membranes with synaptophysin, an abundant protein localized to the synaptic vesicle and a potential regulator of SNARE complex assembly. I showed here that syntaxin/SNAP-25 binding to synaptobrevin in synaptic vesicles causes its dissociation from synaptophysin.Three monoclonal antibodies recognizing the ternary SNARE complex, but not the monomers, were successfully raised and characterised in immunoblots, cell-lines and plasma-membrane sheets. Their binding sites on the complex were mapped. In a functional assay, the antibodies abolished disassembly of the SNARE complex thus providing insights into the disassembly machinery.The antibodies can be used in other functional assays to answer pertinent questions. The status and dynamics of SNARE complexes was probed in pre-synaptic nerve-terminals. I found that the amount of SNARE complex is comparable in resting and stimulated synaptosomes, suggesting that they rapidly disassemble immediately after exocytosis has occurred. Some amount of complex was, however, found on isolated organelles when monitored by direct imaging or immunoblotting, suggesting that rapid disassembly of the SNARE complex is not a pre-requisite for endocytosis, though it cannot be ruled out that complexes formed on isolated organelles in vitro.