The present data demonstrate that the outer membrane of Escherichia coli contains domains of lipopolysaccharide that do not intermix freely with each other. A strain of E. coli lacking galactose epimerase was grown with galactose, for varying periods of time, which permits formation of a long polysaccharide, and without galactose, which results in a short polysaccharide. Such cultures yielded outer membrane fragments that were heterogeneous in lipopolysaccharide composition, some containing more long- than short-chain lipopolysaccharide, and vice versa. The kinetics of formation of these fragments suggest that lipopolysaccharide initially enters the membrane at points from which it can diffuse but ultimately is organized into domains that do not mix with each other, at least when lipopolysaccharides or different composition are present in the same organism.