We report here the identification of a virulence-associated factor, Tcf, (tracheal colonization factor), produced by strains of Bordetella pertussis but not Bordetella parapertussis or Bordetella bronchiseptica. This protein is encoded by the tcfA gene. When a strain of B. pertussis 18323 lacking this protein is used to infect mice with an aerosol challenge, the number of bacteria isolated from the tracheas is decreased 10-fold when compared with the parent 18323. The derived amino acid sequence of tcfA predicts a 68 kDa RGD-containing, proline-rich protein, which after cleavage of a typical prokaryotic signal sequence would be 64 kDa. Amino acid sequence analysis demonstrates that the C-terminal 30 kDa of this protein shows 50% identity to the 30 kDa C-terminus of another Bordetella protein, the pertactin precursor. The N-terminal 34 kDa region contains the three amino-acid motif RGD and is 16.5% proline. Coupled in vitro transcription and translation analysis indicates that the tcfA gene product migrates as two bands of approximately 90 kDa. A fusion protein of the N-terminal, 34 kDa portion of Tcf to maltose-binding protein migrates, on SDS-PAGE, 30 kDa higher than expected from the combined molecular weights. Polyclonal antisera raised against the unique N-terminal portion of Tcf recognizes 90 kDa and 60 kDa bands in immunoblots of whole-cell lysates of strains of B. pertussis; it does not recognize any protein in whole-cell lysates of B. bronchiseptica or B. parapertussis. Supernatants of cultures of B. pertussis 18323 contain the 60 kDa form of the protein. Southern blot analysis of chromosomal DNA from strains of B. bronchiseptica and B. parapertussis, using a probe derived from tcfA, shows strong hybridization only to B. pertussis DNA. Thus, Tcf appears to be a unique virulence factor of B. pertussis.