Protein H, one of the major outer membrane proteins Pseudomonas aeruginosa, shows an interesting interaction with the peptidoglycan layer of the cell. It is retained by peptidoglycan after extraction of the cell envelope with SDS solution at 35 degrees C. A protein of the same molecular weight (21,000) as protein H was found in the peptidoglycan-associated fraction of Escherichia coli K-12 prepared under the same conditions. This protein, designated here as protein 21K, was purified from the cell envelope of E. coli, and the properties of two proteins (protein H and protein 21K) were compared. By gas chromatographic analysis of purified protein 21K and protein H, it was found that both contained covalently linked fatty acid. In isotopic labeling experiments, [14C]palmitic acid and [2-3H]glycerol were incorported into both proteins H and 21K. These two proteins showed similar amino acid compositions, but no apparent correlation was found between protein 21K or protein H and Braun's lipoprotein. These results suggest that protein 21K of E. coli K-12 and protein H of P. aeruginosa PAO are a novel type of lipoprotein. All nine gram-negative bacteria tested, including enteric and non-enteric bacteria, contained a similar protein of apparent molecular weight 21,000 as a peptidoglycan-protein complex.