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We report the novel branching pattern in lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strain 1232. The strain expressed the beta-D-Glcp-(1 -andgt; 4)-[alpha-D-Galp-(1 -andgt; 4)-beta-D-Galp-(1 -andgt; 7)]-D-alpha-D-Hepp-(1 -andgt; 6)-beta-D-Glcp chain linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety of NTHi LPS: L-alpha-D-HepIIIp-(1 -andgt; 2)-[PEtn -andgt; 6]-L-alpha-D-HepIIp-(1 -andgt; 3)-L-alpha-D-HepIp-(1 -andgt; 5)-[PPEtn -andgt; 4]-alpha-Kdop-(2 -andgt; 6)-lipid A. The structure has been elucidated using NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis coupled to electrospray ionization tandem mass spectrometry (CE-ESI-MSn) on O-deacylated LPS and core oligosaccharide (OS) materials, as well as HPLC-ESI-MSo on permethylated, dephosphorylated OS. It was also found that a tetrasaccharide unit bearing sialic acid [alpha-Neu5Ac-(2 -andgt; 3)-beta-D-Galp-(1 -andgt; 4)-beta-D-GlcNAcp-(1 -andgt; 3)-beta-D-Galp-(1 -andgt;] could substitute O-4 of the beta-D-Glcp linked to HepI. In addition, the distal heptose (HepIII) was substituted by PCho -andgt; 6-beta-D-Galp-(1 -andgt; at the O-2 position.