Lipopolysaccharide (LPS), as the major surface molecule of gram-negative bacteria, interacts with the host in complex ways, both inducing and protecting against aspects of inflammatory and adaptive immunity. The membrane-distal repeated carbohydrate structure of LPS, the O antigen, can prevent antibody functions and may vary as a mechanism of immune evasion. Genes of the wbm locus are required for the assembly of O antigen on the animal pathogen Bordetella bronchiseptica and the human pathogen B. parapertussis. However, the important human pathogen B. pertussis lacks these genes and a number of in vitro and in vivo characteristics associated with O antigen in other organisms. To determine the specific functions of O antigen in these closely related Bordetella subspecies, we compared wbm deletion (⌬wbm) mutants of B. bronchiseptica and B. parapertussis in a variety of assays relevant to natural respiratory tract infection. Complement was not activated or depleted by wild-type bordetellae expressing O antigen, but both ⌬wbm mutants activated complement and were highly sensitive to complement-mediated killing in vitro. Although the O-antigen structures appear to be substantially similar, the two mutants differed strikingly in their defects within the respiratory tract. The B. parapertussis ⌬wbm mutant was severely defective in colonization of the tracheas and lungs of mice, while the B. bronchiseptica ⌬wbm mutant showed almost no defect. While in vitro characteristics such as serum resistance may be attributable to O antigen directly, the role of O antigen during infection appears to be more complex, possibly involving factors differing among the closely related bordetellae or different interactions between each one and its host.