Bordetella pertussis, the aetiological agent of whooping cough (pertussis), causes selective destruction of ciliated cells of the human airway mucosa. In a hamster tracheal organ culture model, B. pertussis causes identical cytopathology as does tracheal cytotoxin (TCT), a glycopeptide released by the bacterium. The damage caused by B. pertussis or TCT has been shown to be mediated via nitric oxide (NO·). Using immunofluorescence detection of the cytokine‐inducible NO synthase (iNOS; NOS type II), we determined that B. pertussis induced epithelial NO· production exclusively within non‐ciliated cells. This epithelial iNOS activation could be reproduced by the combination of TCT and endotoxin. However, neither TCT alone nor endotoxin alone was capable of inducing epithelial iNOS. This result mirrors the synergistic activity of TCT and endotoxin exhibited in monolayer cultures of tracheal epithelial cells. Therefore, TCT and endotoxin are both important virulence factors of B. pertussis, combining synergistically to cause the specific epithelial pathology of pertussis.