Haemophilus influenzae is a Gram-negative bacterium that represents a common cause of human disease. Disease due to this organism begins with colonization of the upper respiratory mucosa, a process facilitated by adhesive fibers called pili. In the present study, we investigated the structure and assembly of H. influenzae pili. Examination of pili by electron microscopy using quick-freeze, deep-etch and immunogold techniques revealed the presence of two distinct subassemblies, including a flexible two-stranded helical rod comprised of HifA and a short, thin, distal tip structure containing HifD. Genetic and biochemical studies demonstrated that the biogenesis of H. influenzae pili is dependent on a periplasmic chaperone called HifB, which belongs to the PapD family of immunoglobulin-like chaperones. HifB bound directly to HifA and HifD, forming HifB-HifA and HifB-HifD complexes, which were purified from periplasmic extracts by ion-exchange chromatography. Continued investigation of the biogenesis of H. influenzae pili should provide general insights into organelle development and may suggest novel strategies for disease prevention.