The structure of the O-antigen polysaccharide (PS) from Escherichia coli O74 has been determined. Component analysis, together with (1)H and (13)C NMR spectroscopy as well as (1)H,(15)N-HSQC experiments were employed to elucidate the structure. Inter-residue correlations were determined by (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [structure: see text]. Cross-peaks of low intensity from an alpha-linked N-acetylglucosamine residue were present in the NMR spectra, and spectral analysis indicates that they originate from the penultimate residue in the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-D-glucosamine residue at its reducing end. The (1)H, (13)C and (15)N NMR chemical shifts of the alpha- and beta-anomeric forms of D-Fucp3NAc are also reported. The repeating unit of the E. coli O74 O-antigen is identical to that of the capsular polysaccharide from E. coli K45.