The most consistent pathological feature of pertussis is the selective colonization and subsequent destruction of ciliated cells in the respiratory epithelium. Phase I B. pertussis can reproduce the human respiratory tract cytopathology during in vitro infection of hamster tracheal organ cultures. However, most isolated biologically active components produced by B. pertussis (lymphocytosis-promoting factor, adenylate cyclase, dermonecrotic toxin, etc.) have no apparent cytopathic effect on the respiratory epithelium. We have purified a glycopeptide from the culture supernatant of virulent B. pertussis that mimics completely the ciliated cell pathology characteristic of pertussis (5). Tracheal cytotoxin (TCT) is released during log phase broth culture and consists of 15 amino acid residues as well as two amino sugars. The selective biological activity of TCT has been studied in tracheal organ cultures by light and electron microscopy. A series of pathological changes precedes the eventual extrusion of ciliated cells, while all other epithelial cell types appear ultrastructurally normal. TCT also causes a dose-dependent inhibition of DNA synthesis in cultured hamster trachea epithelial cells, providing a quantitative bioassay to monitor TCT activity during purification steps. Previously, TCT could be completely purified from oxidized glutathione (a major contaminant from the culture medium) only by high-voltage paper electrophoresis, a procedure not well suited for large scale work. We have now substituted a final column chromatography step that separates TCT from oxidized glutathione and other contaminating peptides. This change and other preparative scale adaptations now allow us to purify 150-250 nmol of biologically active TCT from one liter of culture supernatant (3-4 X 10(13) bacteria), a ten-fold increase over our previous batch size with no increase in processing time.(ABSTRACT TRUNCATED AT 250 WORDS)