Botulinum neurotoxin type E, a 150 kDa single chain protein, cleaved with endoproteinase Lys-C yielded 113, 73, and 50 kDa fragments. The N-terminal sequence of the 113 kDa fragment, Gly-Ile-Arg-Lys-Ser-Ile-Cys-Ile, overlaps the N-terminal sequence, Lys-Ser-Ile-Cys-Ile, of the 103 kDa heavy chain produced by nicking the neurotoxin with trypsin. The -Arg-Lys- bond is therefore the site on the single chain type E NT where trypsin nicks generating the 50 kDa light and 103 kDa heavy chains of the dichain NT. The sequence of the first 50 N-terminal residues of the 73 kDa fragment were determined. This fragment is a segment of the heavy chain; 50% of the 50 residues are present in identical positions in a similar segment of the heavy chain of tetanus neurotoxin.