Smooth type endotoxins of Salmonella, Yersinia, and Escherichia were fractionated into long and short chain lipopolysaccharides by silica gel chromatography. Lipid A was prepared from the fractions and analyzed by plasma desorption mass spectrometry. Both Yersinia and Salmonella endotoxins had a large proportion of aminoarabinose-containing lipopolysaccharide molecular species that were found to be concentrated in the long chain fraction. In the Escherichia endotoxin, hypoacylated lipopolysaccharides (lacking the tetradecanoate and one of the four hydroxytetradecanoates) were found mostly in the short chain fraction. Possible implications of these results for the lipopolysaccharide biosynthetic pathway and for studies on the influence of sugar chain length on the biological effects of endotoxins are discussed.