Structural variations in lipid A may alter its pulmonary toxicity. Crude lipid A from Enterobacter agglomerans may contain a number of structurally diverse forms of this toxin. The crude lipid A was obtained by mild acid hydrolysis of E. agglomerans lipopolysaccharide (LPS) in 12-16% yield and was fractionated by silicic acid column chromatography. High performance thin layer chromatography, capillary gas chromatography, and (31)P nuclear magnetic resonance (nmr) spectroscopy were used to characterize several of the largest fractions. Dodecanoic acid, tetradecanoic acid, and hexadecanoic acid were present in approximately equimolar amounts in several of the chromatographic fractions; 3-hydroxytetradecanoic acid was the most abundant fatty acid found in base hydrolyzates of the lipid A fractions. Incorporation of increasing amounts of 2- and 3-hydroxytetradecanoic acid delayed chromatographic elution. Two major peaks were found in (31)P nmr spectra: the resonance at 2.7 ppm was assigned to a glycosidic phosphate monoester (C-1) and that at 4.6 ppm was assigned to a nonglycosidic phosphate monoester (C-4').