Defined mutants of the galactose biosynthetic (Leloir) pathway were employed to investigate lipopolysaccharide (LPS) oligosaccharide expression in Haemophilus influenzae type b strain Eagan. The structures of the low-molecular-mass LPS glycoforms from strains with mutations in the genes that encode galactose epimerase (galE) and galactose kinase (galK) were determined by NMR spectroscopy on O- and N-deacylated and dephosphorylated LPS-backbone, and O-deacylated oligosaccharide samples in conjunction with electrospray mass spectrometric, glycose and methylation analyses. The structural profile of LPS glycoforms from the galK mutant was found to be identical to that of the galactose and glucose-containing Hex5 glycoform previously identified in the parent strain [Masoud, H.; Moxon, E. R.; Martin, A.; Krajcarski, D.; Richards, J. C. Biochemistry1997, 36, 2091-2103]. LPS from the H. influenzae strain bearing mutations in both galK and galE (galE/galK double mutant) was devoid of galactose. In the double mutant, Hex3 and Hex4 glycoforms containing di- and tri-glucan side chains from the central heptose of the triheptosyl inner-core unit were identified as the major glycoforms. The triglucoside chain extension, β-D-Glcp-(1→4)-β-D-Glcp-(1→4)-α-D-Glcp, identified in the Hex4 glycoform has not been previously reported as a structural element of H. influenzae LPS. In the parent strain, it is the galactose-containing trisaccharide, β-d-Galp-(1→4)-β-D-Glcp-(1→4)-α-D-Glcp, and further extended analogues thereof, that substitute the central heptose. When grown in galactose deficient media, the galE single mutant was found to expresses the same population of LPS glycoforms as the double mutant.