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Nontypeable Haemophilus influenzae (NTHi) is an important pathogen responsible for otitis media in children and of pneumonitis in adults with depressed resistance. NTHi is acapsular and, therefore, capsular polysaccharide-based vaccines are ineffective for preventing infections by this pathogen. Recently it was found that a detoxified lipooligo­saccharide (LOS) conjugate from NTHi 9274 induced bactericidal antibodies effective against a large number of NTHi isolates, and conferred protection against NTHi otitis media in chinchillas (X.-X.Gu et al., 1996, Infect. Immun., 64, 4047–4053; X.-X.Gu et al., 1997., Infect. Immun., 65, 4488–4493). In this paper we report the chemical character­ization of the LOS from NTHi 9274 LOS. NTHi is capable of expressing a heterogenous population of LOS exhibited by multiple oligosaccharide (OS) epitopes. OSs released from the LOS of NTHi 9274 by mild acid hydrolysis were purified using Bio-Gel P4 gel permeation chromatography. The OSs were characterized by glycosyl composition analysis, glycosyl linkage analysis, nuclear magnetic resonance spectroscopy (NMR), fast atom bombardment mass spectro­metry (FAB-MS), matrix-assisted laser desorption time of flight mass spectro­metry (MALDITOF-MS), and tandem MS/MS. At least 17 different OS molecules were observed. These contained variable glycosyl residues, phosphate (P), and phospho­ethanolamine (PEA) substituents. These molecules contained either three, four, or five hexoses, and all contained four heptosyl residues. The four heptosyl residues consisted of one d,d-Hep and three l,d-Hep. Dephosphorylation of the OSs with aqueous 48% hydrofluoric acid (HF) reduced the number of molecules to about to seven; Hex1-7Hep4Kdo1. Of these seven, Hex2Hep4Kdo1, Hex3Hep4Kdo1, and Hex4Hep4Kdo1 were the major constituents. Thus, this NTHi LOS preparation is very heterogeneous, and contains structures different from those previously published for Haemophilus influenzae. The tandem MS/MS analysis and glycosyl linkage data suggest that the LOS oligosaccharides have the following structures: [ABSTRACT FROM PUBLISHER]