Gram-negative bacteria are to a large extent covered by lipopolysaccharide (LPS) anchored in the outer leaflet of their outer membrane. There are presently four described pathways for the O-antigen assembly of LPS, viz., synthase-, Wzk-, ABC-transporter- and Wzx/Wzy-dependent pathways, where the latter two are used in Escherichia coli, subject to the O-antigen polysaccharide to be made. NDP-sugar monosaccharides are used by glycosyltransferases in the process of linking sugar residues together in the cytoplasm and depending on the biosynthetic pathway polymerization of the O-antigen takes place either in the cytoplasm (ABC-transporter pathway) or in the periplasm, where an oligosaccharide anchored in the inner membrane is flipped to the periplasmic side to this end (Wzx/Wzy-dependent pathway). Additions of sugars to form side-chains on the O-antigen may also occur in the periplasmic space. The degree of polymerization of the O-antigen is regulated to give a modal distribution, i.e., a narrow distribution around the most probable chain-length. The O-antigen is subsequently conjugated to the lipid A-core to form the LPS, which is transported across the periplasmic region by an ATP-driven mechanism as part of an LPS transport (Lpt) system. By using predictions of NDP-monosaccharide pathways and glycosyltransferase function it is shown how O-antigen structure can be elucidated rapidly by the computer program CASPER using bioinformatics data in conjunction with unassigned NMR data of the polysaccharide.