Pseudomonas stutzeri OXI is a Gram-negative microorganism able to grow in media containing aromatic hydrocarbons. A novel lipo-oligosaccharide from P. stutzeri OX1 was isolated and characterized. For the first time, the presence of two moieties of 4,6-O-(1-carboxy)-ethylidene residues (pyruvic acid) was identified in a core region; these two residues were found to possess different absolute configuration. The structure of the oligosaccharide backbone was determined using either alkaline or acid hydrolysis. Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de-O-acylation) followed by de-N-acylation using hot KOH. The lipo-oligosaccharide was also analyzed after acid treatment, attained by mild hydrolysis with acetic acid, to obtain information on the nature of the phosphate and acyl groups. The two resulting oligosaccharides were isolated by gel permeation chromatography, and investigated by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H-, 31P- and 13C-NMR spectroscopy. These experiments led to the identification of the major oligosaccharide structure representative of core region-lipid A. All sugars are D-pyranoses and α-linked, if not stated otherwise. Based on the structure found, the hypothesis can be advanced that pyruvate residues are used to block elongation of the oligosaccharide chain. This would lead to a less hydrophilic cellular surface, indicating an adaptive response of P. sutzeri OX1 to a hydrocarbon-containing environment.