For investigation of the genes of proteins associated in vivo with botulinum neurotoxin (BoNT), polymerase chain reaction (PCR) experiments were carried out with oligonucleotide primers designed to regions of the nontoxic-nonhemagglutinin (NTNH) gene of Clostridium botulinum type C. The primers were used to amplify a DNA fragment from genomic DNA of C. botulinum types A, B, E, F, G and toxigenic strains of Clostridium barati and Clostridium butyricum. The amplified product from all of these strains hybridized with an internal oligonucleotide probe, whereas all nontoxigenic clostridia tested gave no PCR product and showed no reaction with the probe. The NTNH gene was shown to be located upstream of the gene encoding BoNT, thereby revealing a conserved structure for genes encoding the proteins of the M complex of the progenitor botulinum toxin in these organisms. The sequence of the NTNH gene of nonproteolytic C. botulinum type F was determined by PCR amplification and sequencing of overlapping cloned fragments. NTNH/F showed 71% and 61% identity with NTNH of C. botulinum type E and type C respectively.