The light chain of tetanus neurotoxin (TeNTL chain) has been shown to be endowed with zine endopeptidase activity, selectively directed towards the Gln76-Phe77 bond of synaptobrevin, a vesicle-associated membrane protein critically involved in neuroexocytosis. In previous reports, truncations at the NH2- and COOH-terminus of synaptobrevin have shown that the sequence 39–88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well-defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH2- and COOH-terminal peptides of synaptobrevin, S 27–55 (S1) and S 82–93 (S2), to the synaptobrevin fragment S 56–81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S1 and S2 with complementary sites present on TeNT as shown by surface plasmon resonance experiments. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT probably involving a conformational change of the toxin. This could accound for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins.