Extant Escherichia coli K12 strains are phenotypically rough, their lipopolysaccharide having a complete core structure, but no O antigen. We used DNA hybridization and DNA sequencing to show that the rough phenotype of this strain is due to the presence of one of two independent mutations in the rfb gene cluster. The rfb-50 mutation, consisting of an IS5 insertion at the downstream end of rfb, is present in strain EMG2, which is representative of most K12 derivatives. The rfb-51 mutation is a deletion at the upstream end of rfb, and was found in strain WG1. A gene cloned from strain WG1 could complement the rfb-50 mutation in strain EMG2, and the complemented strain produced O antigen which was typed as O16 with cross reaction to O17.