Abstract Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chains, heavy (H) and light (L). The L chain is a zinc endopeptidase protein highly specific for vesicle-associated membrane protein (VAMP), which is an essential component of the exocytosis apparatus. Here we describe the cloning of the L chain of TeNT from Clostridium tetanistrain Y-IV-3 (WS 15) and its expression in Escherichia colias a glutathione S-transferase fusion protein. The full-length recombinant L chain, corresponding to residues 1–457, was obtained as a mixture of proteins of slightly different mass with identical N-terminal ends. To obtain a product useful for structural analysis and crystallization, a COOH-terminally truncated L chain (residues 1–427) was cloned, expressed, and purified with high yield. This truncated L chain is more active than the full-length and wild-type proteins in the hydrolysis of VAMP. Preliminary experiments of crystallization of the truncated recombinant L chain gave encouraging results.