The formation of small synaptic vesicles represents a hallmark during synaptogenesis. The small synaptic vesicle protein synaptophysin is considered as a marker protein for synapses during neuronal development. Another small synaptic vesicle protein, synaptobrevin, is now well accepted to play an important role for the function of synapses in being a key component of exocytosis. Its role during synaptogenesis is not known. Tetanus toxin which exclusively proteolysis synaptobrevin thereby inhibiting secretion from all types of neurons was used to investigate consequences of inactivating synaptobrevin for the formation of small synaptic vesicles and synaptic contacts. In primary cultures of mouse hypothalamic and cerebellar neurons cultivated for 3 to 4 days, synaptobrevin appears earlier on small synaptic vesicles and in synaptic contacts than synaptophysin. Upon longer cultivation up to 12 to 14 days in vitro both proteins associated equally with small synaptic vesicles. Interestingly, GABA secretion stimulated by 50 mM potassium or 500 PM alpha-latrotoxin, did not vary during cultivation time. Tetanus toxin added to neuronal cultures at day 2 in vitro cleaved synaptobrevin and inhibited regulated GABA secretion during the whole cultivation time. Despite the impaired function of synaptobrevin other synaptic proteins such as synaptophysin, synaptotagmin, rab 3A, protein SV2, SNAP-25 and syntaxin were found in processes and synaptic contacts comparable to untreated cultures. The expression of various synaptic proteins was also followed in vivo. In mouse brains taken at different embryonic days, synaptobrevin, synaptotagmin, rab 6 and the membrane protein SNAP-25 were expressed earlier than synaptophysin and protein SV2. We conclude that synaptobrevin represents a marker for early events in synaptogenesis. Its proteolysis by tetanus toxin, however, does not interfere with the formation of synaptic contacts and neuronal differentiation.