A unique strain of Clostridium botulinum, serotype D 4947 (D-4947), produces a considerable amount of a 650 kDa toxin complex (L-TC) and a small amount of a 280 kDa M-TC, a 540 kDa TC, and a 610 kDa TC. The complexes are composed of only un-nicked components, including neurotoxin (NT), nontoxic nonhemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33 and HA-17). Unlike other NTs from all serotype strains, separation of D-4947 NT from L-TC, except for M-TC, during chromatography required highly alkaline conditions around pH 8.8. The separated NT and NTNHA/HAs complex can be reconstituted to L-TC that is indistinguishable from the parent L-TC with respect to toxicity, hemagglutination activity and gel filtration profile. The isoelectric points of NT and NTNHA/HAs were close together depending on the number of HA-33/17 molecules. We have established a new method to separate the unique D-4947 NT from the complex, which will yield valuable information on structure of botulinum toxin.