A family of high-molecular-weight (HMW) surface-exposed proteins important in the attachment of nontypeable Haemophilus influenzae (NTHi) to human epithelial cells was previously identified (J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875-2879, 1993). In the present investigation, indirect immunogold labeling and electron microscopy were used to localize these proteins on three clinical isolates of NTHi, mutants deficient in expression of one or both HMW proteins, and embedded sections of human oropharyngeal cells after incubation with NTHi strain 12. The filamentous material comprising the proteins was labeled with monoclonal antibodies directed against two prototype HMW proteins (HMW1 and HMW2) of prototype NTHi strain 12. Gold labeling was observed as a cap or discrete aggregate off one pole or centrally along one long axis of the bacterial cell. Heavily labeled, non-bacterial-cell-associated, disk-like aggregates of the HMW proteins were frequently noted in both bacterial preparations as well as in association with the oropharyngeal cell surface and intracellularly. Mutants demonstrated diminished labeling or an absence thereof, respectively, which correlated well with their previously demonstrated reduced ability or inability to adhere to Chang conjunctival epithelial cells in vitro. The Haemophilus HMW proteins share antigenic determinants with and demonstrate amino acid sequence similarity to the filamentous hemagglutinin protein of Bordetella pertussis, a critical adhesin of that organism. The studies presented here demonstrate that the Haemophilus proteins and B. pertussis filamentous hemagglutinin show impressive morphologic and perhaps additional functional similarity.