The biosynthetic function of the lgtABE genetic locus of Neisseria meningitidis was determined by structural analysis of lipopolysaccharide (LPS) derived from mutant strains and enzymic assay for glycosyltransferase activity. LPS was obtained from mutants generated by insertion of antibiotic resistance cassets in each of the three genes lgtA, lgtB, lgtE of the N. meningitidis immunotype L3 strain ϕ3 MC58. LPS from the parent strain expresses the terminal lacto-N-neotetraose structure, Galβ1→4GlcNAcβ1→3Galβ1→4Glc. Mild hydrazine treatment of the LPS afforded O-deacylated samples that were analyzed directly by electrospray ionization mass spectrometry (ESI-MS) in the negative ion mode. In conjunction with results from sugar analysis, ESI-MS revealed successive loss of the sugars Gal, GlcNAc, and Gal in lgt B, lgt A, and lgt E LPS, respectively. The structure of a sample of O- and N-deacylated LPS derived by aqueous KOH treatment of lgt B LPS was determined in detail by two-dimensional homo- and heteronuclear NMR methods. Using a synthetic β-GlcNAc acceptor and a β-lactose acceptor, the glycosyltransferase activities encoded by the lgtB and lgtA genes were unambiguously established. These data provide the first definitive evidence that the three genes encode the respective glycosyltransferases required for biosynthesis of the terminal trisaccharide moiety of the lacto-N-neotetraose structure in Neisseria LPS. From ESI-MS data, it was also determined that the Gal-deficient LPS expressed by the lgt E mutant is identical to that of the major component expressed by immunotype L3 galE-deficient strains. The galE gene which encodes for UDP-glucose-4-epimerase plays an essential role in the incorporation of Gal into meningococcal LPS.