Reference strains of Pseudomonas aeruginosa serogroup O4 (including subgroups O4a,4b, O4a,4c, O4a,4d), serogroup O6, group G, and immunotype 1 are serologically related and produce structurally similar O-specific polysaccharides containing 2-deoxy-2-formamido-D-galacturonic acid, with half of this sugar amidated in group G polysaccharide. We describe the determination of the degree of amidation of this sugar in the other polysaccharides. Each strain's polysaccharide was treated with anhydrous hydrogen fluoride (3 h, 20°) and reduced with sodium borohydride to give oligosaccharide-alditols 3 (acidic) and 4 (neutral). Acidic oligosaccharide 3 (from O4a,4d polysaccharide) was isolated by anion-exchange HPLC (TSK-DEAE column; eluent, 0.03M sodium chloride in 0.01M sodium phosphate buffer, pH 6), and neutral oligosaccharide 4 (from immunotype 1 polysaccharide) was purified by reversed-phase HPLC (Alltech C18 column; eluent, water). The structure of 3 was confirmed by 1H-NMR spectroscopy (sequential selective spin-decoupling and nuclear Overhauser effect experiments), and the structure of 4 was determined by 1H/13C-NMR. The location of the free carboxyl group in 3 and its absence in 4 were proven by pH-dependent NMR spectra (aqueous solutions). Fast-atom-bombardment mass spectrometry confirmed 3 as a monoamide (MW 627) and 4 as a diamide (MW 626). The degree of amidation was estimated by the ratio of oligosaccharides 4 and 3 (measured by anion-exchange/reversed-phase HPLC, monitored by UV absorbance at 220 nm). Results: Immunotype 1 polysaccharide had full amidation of 2-deoxy-2-formamido-D-galacturonic acid; subgroup O4a,4c and serogroup O6 polysaccharides had no amidation; subgroup O4a,4b and O4a,4d had 10% and 20% amidation, respectively; group G polysaccharide had 50% amidation. The possible role of amidation is to adjust the optimal acidity of the lipopolysaccharide layer, with degree varying by strain and likely growing conditions.