ABSTRACTMultiple primer sets were combined with the polymerase chain reaction (PCR) and examined for use in the amplification of toxin gene fragments from four Clostridium botulinum types (A, B, E, and F). Vegetative cells obtained from overnight cultures were used directly in the PCR analyses without purification of chromosomal DNA. Gene fragments were amplified from the different botulinal toxin genes that code for types A, B, E and F toxins using a single PCR protocol. Toxin gene fragments were amplified from types B and F toxigenic organisms using the PCR and specific primer sets for these types in a single PCR tube. Type A and E toxin genes were also examined using type A and E specific primers in a separate PCR tube. The PCR‐amplified products were separated by electrophoresis on agarose gels containing ethidium bromide. The identity of the PCR products were confirmed by DNA hybridization using type specific probes. We conclude that this method is useful for the rapid and direct identification of toxigenic botulinal organisms that code for the toxin types A, B, E, or