Structures for the N-acetylneuraminic acid (Neu5Ac)-containing O56 and O24 polysaccharides of Escherichia coli have been reported previously. During these studies unusual chemical shifts had been observed for the NMR signals for H-3eq and C-3 of the Neu5Ac residues of both polysaccharides. In further pursuing this phenomenon, we have reinvestigated the O56 and O24 polysaccharides as well as derived oligosaccharides by one- and two-dimensional NMR spectroscopy. The results showed that structures of both polysaccharides (PSs) had to be modified and formulated as2D ROESY spectra revealed a strong NOE between H-3eq of Neu5Ac and the protons of the side-chain sugar (H-3 and H-5 of α-d-Galp in the O56 PS and H-3 of α-d-Glcp in the O24 PS) and also between H-3ax of Neu5Ac and H-3 of β-d-Glcp in the main chain. This indicated a close spatial apposition of the seven-linked α-Neu5Ac and the side-chain residues α-d-Galp (O56 PS) and α-d-Glcp (O24 PS), respectively. The strong long-range spatial contacts caused the unusual chemical shifts of H-3eq and C-3 of Neu5Ac.