Botulinum neurotoxin (NT) serotype A is a approximately 150-kDa dichain protein. Posttranslational nicking of the single-chain NT (residues Pro 1-Leu 1295) by the protease(s) endogenous to Clostridium botulinum excises 10 residues, leaving Pro 1-Lys 437 and Ala 448-Leu 1295 in the approximately 50-kDa light (L) and approximately 100-kDa heavy (H) chains, respectively, connected by a Cys 429-Cys 453 disulfide and noncovalent bonds [Krieglstein et al. (1994), J. Protein Chem. 13, 49-57]. The L chain is a metalloprotease, while the amino- and carboxy-terminal halves of the H chain have channel-forming and receptor-binding activities, respectively [Montecucco and Schiavo (1995), Q. Rev. Biophys. 28, 423-472]. Endoproteinase Glu-C and alpha-chymotrypsin were used for controlled digestion at pH 7.4 of the approximately 150-kDa dichain NT and the isolated approximately 100-kDa H chain (i.e., freed from the L chain) in order to map the cleavage sites and isolate the proteolytic fragments. The dichain NT appeared more resistant to cleavage by endoproteinase Glu-C than the isolated H chain. In contrast, the NT with its disulfide(s) reduced showed rapid digestion of both chains, including a cleavage between Glu 251 and Met 252 (resulting in approximately 30- and approximately 20-kDa fragments of the L chain) which was not noted unless the NT was reduced. Interestingly, an adjacent bond, Tyr 249-Tyr 250, was noted earlier [DasGupta and Foley (1989), Biochimie 71, 1193-1200] to undergo "self-cleavage" following reductive separation of the L chain from the H chain. The site Tyr-Tyr-Glu-Met (residues 249-252) appears to become exposed following reduction of Cys 429-Cys 453 disulfide. Identification of Glu 669-Ile 670 and Tyr 683-Ile 684 as protease-susceptible sites demonstrated for the first time that at least two peptide bonds in the segment of the H chain (residues 659-684), part of which (residues 659-681) is thought to interact with the endosomal membranes and forms channels [Oblatt-Montal et al., (1995), Protein Sci, 4, 1490-1497], are exposed on the surface of the NT. Two of the fragments of the H chain we generated and purified by chromatography are suitable for structure-function studies; the approximately 85- and approximately 45-kDa fragments beginning at residue Leu 544 and Ser 884, respectively (both extend presumably to Leu 1295) contain the channel-forming segment and receptor-binding segments, respectively. In determining partial amino acid sequences of 10 fragments, a total of 149 amino acids in the 1275-residue NT were chemically identified.