Lipopolysaccharides from Pseudomonas aeruginosa O1 (Lányi classification), O3 (Habs classification), O13 and O14 (Wokatsch classification), and strain NCTC 8505, which is also related to serogroup O3 (Habs), have structurally similar O-specific polysaccharide chains built up of tetrasaccharide repeating units involving L-rhamnose (Rha), 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-galacturonic acid (GalNAcA), and a di-N-acyl derivative of bacillosamine (BacN): 2,4-diacetamido-2,4,6-trideoxy-D-glucose or 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido]-D-glucose. The latter derivative was obtained free by solvolysis with hydrogen fluoride of carboxyl-reduced Habs O3 polysaccharide, and was identified by 1H-nuclear magnetic resonance spectroscopy and by mass spectrometry of the corresponding methylated alditol. Habs O3, Lányi O1, and Wokatsch O14 polysaccharides contained O-acetyl groups. Solvolysis with hydrogen fluoride of the native Habs O3 polysaccharide resulted in selective cleavage of the glycosidic linkages of 6-deoxy sugars to give the trisaccharide fragment involving all three N-acylated amino sugars. Similar solvolysis of NCTC 8505 polysaccharide afforded a mixture of disaccharide and trisaccharide with N,N'-diacetylbacillosamine at the reducing end. Smith degradation of Habs O3 polysaccharide resulted in selective oxidation of rhamnose to give a glycoside of a trisaccharide with glyceraldehyde as the aglycone. Smith degradation of NCTC 8505 polysaccharide was complicated by the formation of the glycoside of a trisaccharide with an aglycone of unknown structure. A trisaccharide with rhamnose at the reducing end was also isolated after Smith degradation of the latter polysaccharide. Analysis of the composition and structure of all oligosaccharides obtained, and detailed examination of the 13C-nuclear magnetic resonance spectra of these oligosaccharides, and of both intact and modified polysaccharides, revealed the following structures of the repeating units. The structure for the NCTC 8505 polysaccharide differs from that proposed previously [Tahara, Y. and Wilkinson, S.G. (1983) Eur. J. Biochem. 134, 299-304] in the configurations assigned to the glycosidic linkages of rhamnose and bacillosamine. The results obtained show the P. aeruginosa strains studied to represent three different O-serotypes in a single O-serogroup (Formula: see text).