Bordetella pertussis is the only bacteria] species which is known to produce pertussis toxin (PT); however, both Bordetella bronchiseptica and Bordetella parapertussis contain regions homologous to the ptx genes of B. pertussis that encode the toxin subunits. After finding that several children with B. parapertussis infections exhibited modest antibody titers to PT, we examined the ptx genes of both B. parapertussis and B. bronchiseptica to determine whether they would encode stable, functional proteins even though their promoters are thought to be inactive under the conditions that have been examined. We inserted a functional promoter directly upstream of the ptx-ptl region of both species and examined culture supernatants of the resulting strains for PT activity. Biologically active PT was found in the culture supernatants of both engineered species. The toxin encoded by the B. parapertussis ptx genes appeared more labile in culture supernatants than did toxin produced by either B. pertussis or the engineered strain of B. bronchiseptica. This lability might be due to the lack of a full-length S2 subunit. We also investigated the ptl genes of these species, which are necessary for the secretion of this toxin, and found that both B. bronchiseptica and B. parapertussis contain at least certain of these genes, including ptlE and ptlF. Moreover, B. bronchiseptica appeared to contain all essential ptl genes since the introduction of a functional promoter directly upstream of the ptx-ptl region resulted in both production and efficient secretion of toxin. These results indicate that despite a number of amino acid changes in the sequences of the toxins, the toxins encoded by B. bronchiseptica and B. parapertussis are active.