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All members of the genus Bordetella and Pasteurella multocida (a gram-negative bacillus genetically unrelated to Bordetella spp., yet often sharing the same ecological niche) produce a dermonecrotic toxin (DNT). The amount of toxin produced and the time required for appearance of the lesions are identical for Bordetella pertussis, B. parapertussis, and B. bronchiseptica but different for P. multocida and B. avium. DNT has been reported to act by promoting vasoconstriction; however, vasoactive compounds (verapamil, prazosin, hydralazine, tolazoline, or isoxsuprine) are able to reverse the action of the toxin only slightly. Vasoconstrictors (atropine, serotonin, epinephrine, or endothelin) did not produce DNT-like lesions. We have characterized a region of DNA essential for DNT expression. We have determined by Southern analysis that the restriction map of the DNT gene is nearly identical in B. pertussis, B. parapertussis, and B. bronchiseptica, but the sequences are not present in toxigenic B. avium and P. multocida strains. A gentamicin resistance-origin of transfer cassette cloned into a 1.8-kb NotI-BamHI fragment results in constructs which can be mobilized and recombined into the Bordetella chromosome, rendering the resultant B. pertussis, B. parapertussis, and B. bronchiseptica strains negative for DNT. A 5-kb BamHI-ApaI fragment from the B. pertussis chromosome was sequenced and revealed homology to the Escherichia coli CNF1 (cytotoxic necrotizing factor 1) toxin.