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Lipopolysaccharide from Escherichiu coli 01 11, its gulE derivative when grown in galactose, E. coli 086, and Sulmonella typhimuvium LT2 all contain antigenic side chains and separate into more than 40 components by electrophoresis in gradients of polyacrylaniide containing sodium dodecylsulfate. These components from E. coli 0111 are not interconvertible and show a heterogeneous size distribution when fractionated with Sephadex (3-200. Isoelectric focusing of this mixture in pH 3.5 -10 ampholines reveals a single component, ruling out extensive charge heterogeneity.The relative antigenic side chain lengths for the components, estimated using ratios of galactose in antigenic side chain to phosphate in the lipid-A-core oligosaccharide region, show that the size heterogeneity is due to differences in the number of antigenic side chain units per molecule and ranges from none to over 40. Preference for molecules of specific chain lengths, especially short ones, was observed.In contrast, the galE mutant grown without galactose does not synthesize antigenic side chains, and more than 90 % of its lipopolysaccharide migrates as a single band at a position corresponding to the lowest-molecular-weight component from the above preparations. Lipopolysaccharide from E. coli PL2, a K12 strain lacking antigenic side chain, separates into two low-molecular-weight components on electrophoresis. These results confirm that the heterogeneity which we observe in lipopolysaccharide containing antigenic side chains, is due to the side chain rather than the lipid-A -core oligosaccharide region.Lipopolysaccharide is found uniquely in the outer membrane of gram-negative bacteria and is important in the structure [ 1,2] and function [3,4] of this membrane. Variations in its composition also influence host responses to invasion by gram-negative organisms [5].Lipopolysaccharide consists of lipid A, core oligosaccharide, and antigenic side chain. The linkage of sugar residues [6] and the chemical structure within the lipid A moiety [7,8] is known for lipopolysaccharide from several organisms. Biosynthesis of lipid A has also been investigated [9].