Outer membrane proteins O-8 and O-9 have been highly purified from a strain of Escherichia coli K-12 by Sephadex G-200 and DEAE-cellulose chromatographies. The amino acid compositions of the purified proteins were definitely different, although they showed marked similarities. The profiles of BrCN peptides of the two proteins were also different. None of the BrCN peptides were the same for the two proteins. Analysis of the first twelve N-terminal residues revealed that the two proteins are strikingly similar, but with differences in the third and the eleventh amino acid residues. It can be concluded that proteins O-8 and O-9 are products of different structural genes which developed by duplication of an ancestral genome followed by mutation.