Synaptophysin and synaptobrevin/VAMP are abundant synaptic vesicle proteins that form homo- and heterooligomers. We now use chemical cross-linking in synaptosomes, pinched-off nerve terminals that are capable of stimulus-dependent neurotransmitter release, to investigate whether these complexes are regulated. We show that in synaptosomes treated with three stimuli that induce exocytosis (a depolarizing K(+) solution, the excitatory neurotoxin alpha-latrotoxin, or the Ca(2+)-ionophore ionomycin), the homo- and heteromultimerization of synaptophysin and synaptobrevin is increased up to 6-fold. Whereas at rest less than 10% of the total synaptobrevin and synaptophysin could be chemically cross-linked into homo- and heteromeric complexes, after stimulation up to 25% of synaptobrevin and synaptophysin are present in homo- and heteromultimers, suggesting that a large fraction of these synaptic vesicle proteins physiologically participate in such complexes. The increase in multimerization of synaptophysin and synaptobrevin was only observed in intact but not in lysed synaptosomes and could not be inhibited by general kinase or phosphatase inhibitors. The stimulus dependence of synaptophysin and synaptobrevin multimers indicates that the complexes are not composed of a fixed multisubunit structure, for example, as an ion channel, but represent distinct functional states of synaptobrevin and synaptophysin that are modulated in parallel with synaptic vesicle exo- and endocytosis.