Tetanus toxin (TeTx) and the various forms of botulinal neurotoxins (BoNT/A to BoNT/G) potently inhibit neurotransmission by means of their L chains which selectively proteolyze synaptic proteins such as synaptobrevin (TeTx, BoNT/B, BoNT/F), SNAP-25 (BoNT/A), and syntaxin (BoNT/C1). Here we show that BoNT/D cleaves rat synaptobrevin 1 and 2 in toxified synaptosomes and in isolated vesicles. In contrast, synaptobrevin 1, as generated by in vitro translation, is only a poor substrate for BoNT/D, whereas this species is cleaved by BoNT/F with similar potency. Cleavage by BoNT/D occurs at the peptide bond Lys59-Leu60 which is adjacent to the BoNT/F cleavage site (Gln58-Lys59) and again differs from the site hydrolyzed by TeTx and BoNT/B (Gln76-Phe77). Cellubrevin, a recently discovered isoform expressed outside the nervous system, is efficiently cleaved by all three toxins examined. For further characterization of the substrate requirements of BoNT/D, we tested amino- and carboxyl-terminal deletion mutants of synaptobrevin 2 as well as synthetic peptides. Shorter peptides containing up to 15 amino acids on either side of the cleavage site were not cleaved, and a peptide extending from Arg47 to Thr116 was a poor substrate for all three toxins tested. However, cleavability was restored when the peptide is further extended at the NH2 terminus (Thr27-Thr116) demonstrating that NH2 terminally located sequences of synaptobrevin which are distal from the respective cleavage sites are required for proteolysis. To further examine the isoform specificity, several mutants of rat synaptobrevin 2 were generated in which individual amino acids were replaced with those found in rat synaptobrevin 1. We show that a Met46 to Ile46 substitution drastically diminishes cleavability by BoNT/D and that the presence of Val76 instead of Gln76 dictates the reduced cleavability of synaptobrevin isoforms by TeTx.