The structure of the O-antigen polysaccharide (PS) from Escherichia coli O177 has been determined. Component analysis together with (1)H and (13)C NMR spectroscopy experiments was used to determine the structure. Inter-residue correlations were determined by (1)H,(13)C-heteronuclear multiple-bond correlation and (1)H,(1)H-NOESY experiments. PS is composed of tetrasaccharide repeating units with the following structure: →2)-α-L-Rhap-(1→3)-α-L-FucpNAc-(1→3)-α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→. An α-L-Rhap residue is suggested to be present at the terminal part of the polysaccharide, which on average is composed of ∼20 repeating units, since the (1)H and (13)C chemical shifts of an α-linked rhamnopyranosyl group could be assigned by a combination of 2D NMR spectra. Consequently, the biological repeating unit has a 3-substituted N-acetyl-D-glucosamine residue at its reducing end. The repeating unit of the E. coli O177 O-antigen shares the →3)-α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→ structural element with the O-antigen from E. coli O15 and this identity may then explain the reported cross-reactivity between the strains.