Haemophilus influenzae is a gram-negative pleiomorphic bacterium that is a common commensal/mutualist within the human airways (30). Encapsulated H. influenzae strains are overt pathogens causing invasive disease (3) and have largely been contained by a vaccine effective against the predominant capsular serotype b strains (32). In contrast, the so-called nontypeable H. influenzae (NTHi) strains lacking capsular polysaccharides remain predominant in asymptomatic carriage and localized airway infections (14, 29). These infections are mostly opportunistic in nature and include bronchiopneumonia, sinusitis, and otitis media (OM). OM is among the most common pediatric infections, causing an estimated ϳ$5 billion in costs of treatment and parents' missed work days per year (20). OM infections include chronic OM that is difficult to resolve with antibiotic therapy, and it has long been postulated that chronic OM involves the formation of bacterial biofilm communities (5, 35). In support of that hypothesis, biofilms have been visualized in tympanostomy drain tubes removed from patients with OM and on middle ear tissue from experimentally infected chinchillas (7,18,33). More recent evidence shows that NTHi and other bacterial agents are present within biofilms on tissue specimens obtained from patients with chronic and recurrent OM (13).The H. influenzae surface is covered with lipooligosaccharide (LOS) endotoxins that lack a repeating O side chain. Instead, the H. influenzae LOS features a diverse collection of LOS glycoforms that differ in the length, content, and nature of the chemical linkages found in the oligosaccharide portion. These LOS oligosaccharides include structures that are antigenically similar to host cell-surface glycolipids and may also contain the host membrane constituents sialic acid (NeuAc) and phosphorylcholine (PCho) (41). LOS confers resistance to host killing (8,9,37) and is also the primary target of the Toll-like receptor 4 pathway that mediates protection against H. influenzae in the airways (47). It has been established that NTHi strains that express NeuAc-LOS forms comprise a greater proportion of biofilm communities than of planktonic cultures, and that mutations eliminating these forms decrease biofilm formation and bacterial persistence in animal models of OM (4,12,18,43). More recently, we showed that LOS purified from biofilms has decreased potency as an inflammatory agonist, which correlated with an increase in PCho ϩ LOS forms that were present within biofilms (55). In this study, we compared the virulence