Non-typeable Haemophilus influenzae (NTHi) are opportunistic mucosal pathogens which adhere to epithelial cells via a variety of non-specific and specific interactions. Several adhesins have been identified and while the complimentary receptor(s) for each of these adhesins has not yet been fully characterized, it is widely accepted that adherence is an absolute prerequisite for disease. Several reports have indicated that NTHi can also be internalized and reside intracellularly. For this to occur, NTHi must be taken up by mucosal epithelial cells lining the respiratory tract. We have noted, by TEM, that adherent NTHi overlie an electron dense area in the cell membrane of human epithelial cells which is associated with a localized complex assembly of cytoskeletal fibers in the eukaryotic cytoplasm. We thus examined the potential involvement of cytoskeletal actin in this phenomenon via FITC-phalloidin labeling of respiratory tract epithelial cells which had been incubated with several clinical isolates of NTHi. Strong punctate fluorescence was coincident with adherent NTHi to both human oropharyngeal and chinchilla middle ear epithelial cells. This reactivity was similar to the discrete fluorescent spots observed with enteropathogenic Escherichia coli which were adhered to HeLa cells. In contrast, none of the NTHi isolates tested induced actin polymerization in cells of endothelial origin. While the exact mechanisms involved are yet to be elucidated, our data indicated that actin nucleation was coincident with NTHi adherence.