Ribosomal protein S6 of wild-type strains of Escherichia coli contains up to six glutamic acid residues at its C-terminus. The first two residues are encoded by the structural gene for this protein (rpsF) and the rest are added post-translationally. Mutants deficient in this modification were isolated and characterized genetically and biochemically. The S6 protein in these mutants appeared to contain only two glutamic acid residues at the C-terminus as expected. The mutated gene was termed rimK and was mapped at 18.7 min between cmlA and aroA. The rimK gene was cloned into a cosmid vector and its nucleotide sequence determined. Analysis of the transcriptional and translational products of this gene indicates that it encodes a protein with an Mr of 31.5 kDa and that it forms an operon with a gene encoding a 24 kDa protein. An rpsF mutant containing a Glu to Lys replacement in the second residue from the C-terminus of protein S6 was isolated. The S6 protein of this mutant was apparently inaccessible to the RimK modification system. This indicates that the RimK modification system requires the wild-type amino acid sequence at least in the C-terminal region of ribosomal protein S6.