Lipopolysaccharide (LPS) from E. coli K-12 W3100 is known to contain several glycoforms and the basic structure has been investigated previously by methylation analyses (Holst, O. (1999) In Endotoxin in Health and Disease, Brade, H., Opal, S.M., Vogel, S.N., & Morrison, D.C., eds, pp. 115-154; Marcel Dekker, Inc., New York). In order to reveal dependencies of gene activity and LPS structure, we have now determined the composition of de-O-acylated LPS by ESI-FT-MS and identified eleven different LPS molecules. We have isolated the major glycoforms after de-O- and de-N-acylation and obtained four oligosaccharides which differed in their carbohydrate structure and phosphate substitution. The main oligosaccharide accounted for appr. 70% of the total and had a molecular weight of 2516 Da according to ESI-FT-MS. The dodecasaccharide structure (glycoform I) as determined by NMR was consistent with MS and compositional analysis. One minor oligosaccharide (5%) of the same carbohydrate structure did not contain the 4' phosphate of the lipid A. Two oligosaccharides contained the same phosphate substitution but differed in their carbohydrate structure, one (5%) which contained an additional -D-GlcN in (1-7)-linkage on a terminal heptose residue (glycoform II) which was N-acteylated in LPS. A minor amount of a molecule lacking the terminal L--D-Hep in the outer core but otherwise identical to the major oligosaccharide (glycoform III) could only be identified by ESI-FT-MS of the de-O-acylated LPS. The other oligosaccharide (20%) contained an -Kdo-(2-4)-[-L-Rha-(1-5)]--Kdo-(2-4)--Kdo branched tetrasaccharide connected to the lipid A (glycoform IV). This novel inner core structure was accompanied by a truncation of the outer core in which the terminal disaccharide L--D-Hep-(1-6)--D-Glc was missing. The latter structure was identified for the first time in LPS and revealed that changes in the inner core structure may be accompanied by structural changes in the outer core.