The O-specific polysaccharides of Pseudomonas aeruginosa 0:3 (Lanyi classification) possess similar structures composed of trisaccharide repeating-units containing 2-acetamido-2-deoxy-D-fucose and uncommon acidic diamino sugars. Recently, two of these sugars were identified as 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid and its 2-imidazoline derivative. We now report the identification of 2,3-diacetamido-2,3-dideoxy-L-guluronic acid as a constituent of P. aeruginosa 0:3a,d,e (strain 170006) O-specific polysaccharide. The acidic polysaccharide was obtained by mild acid degradation (1% CH3CO2H, 100°C, 2 h) of the lipopolysaccharide isolated from dry bacterial cells via the Westphal procedure. Hydrolysis (4M HCl, 100°C, 4 h) followed by sugar analysis, 13C-NMR spectroscopy, solvolysis with hydrogen fluoride (25°C, 3 h) to obtain the acidic trisaccharide (chemical repeating-unit), sequential treatment with borohydride, periodate, borohydride, and 5% aqueous triethylamine (60°C, 3 h) to cleave the imidazoline ring, carboxyl-reduction of the resulting oligosaccharide, acetylation, and comparison of 360-MHz 1H-NMR (coupling constants: J1'2' 1.5, J2'3' 3.6, J3'4' 10.0, J4'5' 10.0 Hz) and 13C-NMR spectra with analogous oligosaccharides from P. aeruginosa 0:3a,d O-specific polysaccharide were performed. These analyses proved the configuration of the terminal hexose residue as manno, assigned signals for the penultimate hexose residue, and confirmed the presence of the new sugar. Thus, the O-specific polysaccharide of P. aeruginosa 0:3a,d,e contains 2,3-diacetamido-2,3-dideoxy-L-guluronic acid, which has not been observed previously in Nature, and the structure of its chemical repeating-unit is the trisaccharide 1.