On mild acid degradation of the lipopolysaccharide of Escherichia coli O108, the O-polysaccharide was isolated and studied by sugar analysis and one- and two-dimensional 1H- and 13C-NMR spectroscopy. The polysaccharide was found to contain an unusual higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyl-8-epilegionaminic acid, 8eLeg5Ac7Ac). The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: -->4)-alpha-8eLegp5Ac7Ac-(2-->6)-alpha-D-Galp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D-GlcpNAc-(1-->. Functions of the E. coli O108 antigen biosynthetic genes, including seven putative genes for synthesis of 8eLeg5Ac7Ac, were assigned by sequencing the O-antigen gene cluster along with comparison with gene databases and known biosynthetic pathways for related nonulosonic acids.