<jats:title>Abstract</jats:title><jats:p>A 9.8‐kbp DNA fragment which contained a neurotoxin gene and its upstream region was cloned from <jats:italic>Clostridium botulinum</jats:italic> type D strain CB‐16. Nucleotide sequencing of the fragment revealed that genes encoding for hemagglutinin (HA) subcomponents and one for a nontoxic‐nonhemagglutinin (NTNH) component were located upstream of the neurotoxin gene. This strain produced two toxins of different molecular size (approximately 300 kDa and 500 kDa) which were designated as progenitor toxins (M and L toxins). The molecular size of the NTNH component of L toxin was approximately 130 kDa on SDS‐PAGE and its N‐terminal amino acid sequence was M‐D‐I‐N‐D‐D‐L‐N‐I‐N‐S‐P‐V‐D‐N‐K‐N‐V‐V‐I which agreed with that deduced from the nucleotide sequence. In contrast, the M toxin had a 115‐kDa NTNH component whose N‐terminal sequence was S‐T‐I‐P‐F‐P‐F‐G‐G‐Y‐R‐E‐T‐N‐Y‐I‐E, corresponding to the sequence from Ser141 of the deduced sequence. A 15‐kDa fragment, which was found to be associated with an M toxin preparation, possessed the same N‐terminal amino acid sequence as that of the 130‐kDa NTNH component. Furthermore, five major fragments generated by limited proteolysis with V8 protease were shown to have N‐terminal amino acid sequences identical to those deduced from the nucleotide sequence of 130‐kDa NTNH. These results indicate that the 130‐kDa NTNH of the L toxin is cleaved at a unique site, between Thr and Ser, leading to the 115‐kDa NTNH of the M toxin.</jats:p>