To successfully colonize their mammalian hosts, many bacteria produce multiple virulence factors that play essential roles in disease processes and pathogenesis. Some of these molecules are adhesins that allow efficient attachment to host cells, a prerequisite for successful host colonization. Bordetella spp. express a number of proteins which either play a direct role in attachment to the respiratory epithelia or exhibit similarity to known bacterial adhesins. One such recently identified protein is BipA. Despite the similarity of BipA to intimins and invasins, deletion of this protein from B. bronchiseptica did not result in any significant defect in respiratory tract colonization. In this study, we identified an open reading frame in B. bronchiseptica, designated bcfA (encoding BcfA [bordetella colonization factor A]), that is similar to bipA. In contrast to the maximal expression of bipA in the Bvg intermediate (Bvg(i)) phase, bcfA is expressed at high levels in both the Bvg(+) and Bvg(i) phases. We show here that BvgA and phosphorylated BvgA bind differentially to the bcfA promoter region. Utilizing immunoblot assays, we found that BcfA is localized to the outer membrane and that it is expressed during animal infection. While deletion of either bipA or bcfA did not significantly affect respiratory tract colonization, concomitant deletion of both genes resulted in a defect in colonization of the rat trachea. Our results indicate that the two paralogous proteins have a combinatorial role in mediating efficient respiratory tract colonization.