SummaryHemophilusinfluenzae type b is a Gram‐negative bacillus that initiates infection by colonizing the upper respiratory tract. Previous studies have established that H. influenzae haemagglutinating pili possess adhesive properties and influence the process of colonization. Additional studies suggest the presence of a secondH. influenzaeadhesin distinct from haemagglulinating pili. In the present study, we examined a non‐piliated H.influenzaetype b strain by transmission electron microscopy and visualized occasional short, thin, surface fibrils. Subsequently, we isolated a spontaneous mutant that lacked surface fibrils and was deficient in adherence to cultured human epithelial cells. Using a cloning strategy that exploited this mutant, we isolated a fragment of DNA that promotesin vitroadherence to human epithelial cells when expressed in laboratory strains of Escherichia coli. Mutagenesis of this fragment in a series ofH. influenzaetype b strains resulted in loss of expression of surface fibrils and a marked decrease in attachment. Furthermore, restoration of surface fibril expression was associated with reacquisition of wild‐type levels of adherence. Our results are consistent with the conclusion thatH. influenzaetype b surface fibrils have adhesive capacity. We speculate that these organelles facilitate colonization of the human respiratory