Mucin-specific adhesion of Pseudomonas aeruginosa plays an important role in the initial colonization of this organism in the airways of cystic fibrosis patients. We report here that the flagellar cap protein, FliD, participates in this adhesion process. A polar chromosomal insertional mutation in the P. aeruginosa fliD gene made this organism nonadhesive to mucin in an in vitro mucin adhesion assay. The adhesive phenotype was restored by providing the fliD gene alone on a multicopy plasmid, suggesting involvement of this gene in mucin adhesion of P. aeruginosa. Further supporting this observation, the in vitro competition experiments demonstrated that purified FliD protein inhibited the mucin adhesion of nonpiliated P. aeruginosa PAK-NP, while the same concentrations of PilA and FlaG proteins of P. aeruginosa were ineffective in this function. The regulation of the fliD gene was studied and was found to be unique in that the transcription of the fliD gene was independent of the flagellar sigma factor sigma28. Consistent with this finding, no sigma28 binding sequence could be identified in the fliD promoter region. The results of the beta-galactosidase assays suggest that the fliD gene in P. aeruginosa is regulated by the newly described transcriptional regulator FleQ and the alternate sigma factor sigma54 (RpoN).