A large fraction of a constitutively synthesized polypeptide, comprising 5% of the total Escherichia coli protein, is released when plasmolysed cells are subjected to osmotic shock into ice-cold water. Since the protein is not liberated by the conversion of cells to spheroplasts, it is not a typical periplasmic protein. A complex pattern of association with the cell envelope indicates that it is bound to this structure in vivo. Its susceptibility to trypsin and its interaction with specific antibodies vary with the type of preparations used. Based on these observations, we postulate a peripheral location at the inner surface of the plasma membrane. The protein has been purified to homogeneity from osmotic shock fluid. It has a mass of 44 000 daltons. Some of its physical and chemical properties have been investigated. Most remarkable are its strongly aggregating and adhesive characteristics and its precipitation by vinblastine and calcium ions. These unusual properties, its presumed location, and the observation that it is present in large amounts (approximately 70 000 molecules per cell) suggest a structural role for this protein.