Differences between the type B neurotoxin gene sequence of Clostridium botulinum type A(B) and Cl. botulinum type B, including a six nucleotide deletion, were recently proposed as a cause of the lack of expression of this gene in the type A toxigenic strains. A polymerase chain reaction (PCR) based on two sets of primers was designed to investigate the absence of the 6-nucleotide sequence in the apparently unexpressed type B toxin gene of 42 strains of Cl. botulinum type A(B). Thirty-five strains were shown to exhibit a deletion in their type B toxin gene; two strains did not have the deletion and actually produced small amounts of type B toxin when tested by the mouse bioassay. This two-step PCR might be useful for the rapid determination of the presence of the six nucleotide deletion and consequently, whether the type B toxin is likely to be produced.