Tetanus toxin is synthesized by Clostridium tetani as a 151-kDa peptide chain. The primary gene product is processed post-translationally by removal of the initiating methionine residue, formation of disulfide bridges and limited proteolysis by bacterial or exogenous proteinases. The mature toxins consist of a 52-kDa light chain and a 98-kDa heavy chain, linked together by a disulfide bond. Proteolytic nicking is accompanied by increased pharmacological potency. To identify the structural alterations involved, single-chain toxin has been subjected to limited proteolysis with various enzymes. The new N-termini have been determined by Edman degradation and the C-termini by isolation of short C-terminal peptide fragments and subsequent analysis of the sequence and composition. All two-chain toxins result from proteolytic nicking within the 17-residue segment of residues 445-461. Thus, the protease(s) of the culture broth cleave on the C-terminal side of Glu449 and partially Ala456, giving rise to two heavy chain N-termini. Trypsin and clostripain first attack the C-terminal of Arg454 and later Arg448, whereas endoproteinase Arg-C cleaves the former bond only. Chymotrypsin and endoproteinase Glu-C each split a single peptide bond, i.e. that located after Tyr452 and Glu449, respectively. Papain gives rise to a large number of cleavages within the 17-residue segment, the new C-terminus being Thr445 or Asn446 and the new N-terminus being Asp460 or Leu461. Further papain digestion leads to an additional cleavage within the heavy chain between Ser863 and Lys864. The original N-terminal Pro1 and C-terminal Asp1314, predicted from the nucleotide sequence, are conserved in all proteolytic digests. The pharmacological activity of the various two-chain toxins was 5-11 times that of the single-chain toxin, as estimated from the inhibition of [3H]noradrenaline release from rat-brain homogenate. The present data on the processing and activation by limited proteolysis prove the existence of several active tetanus isotoxins. These data, together with our previous data on the localization of disulfide bridges and sulfhydryl groups (Krieglstein, K., Henschen, A., Weller, U. & Habermann, E. (1990) Eur. J. Biochem. 188, 39-45), provide the detailed protein chemical characterization of the tetanus isotoxins.