Microcin C51 (MccC51) is an antimicrobial nucleotide-heptapeptide produced by a natural Escherichia coli strain. A 5.7-kb fragment of the pC51 plasmid carrying the genes involved in MccC51 production, secretion, and self-immunity was sequenced, and the genes were characterized. The sequence of the MccC51 gene cluster is highly similar to that of the MccC7 gene. Recombinant plasmids carrying different combinations of the mcc genes involved in the MccC51 production or immunity were constructed to characterize their functional roles. The mccA, mccB, mccD, and mccE genes are involved in MccC51 production, while the mccC and mccE genes are responsible for immunity to MccC51. The mcc gene cluster is flanked by 44-bp direct repeats. Amino acid sequence comparisons allowed us to propose functions for each Mcc polypeptide in MccC51 biosynthesis. Plasmid pUHN containing the cloned mccA, mccB, mccC, and mccE genes, but lacking mccD, directed the synthesis of MccC51p, a substance chemically related to MccC51. MccC51p exhibited weak antibiotic activity against E. coli and was toxic to the producing cells. The immunity to exogenous MccC51 determined by the mccC and mccE genes did not overcome the toxic action of MccC51p on the producing cells. The G+C content of the MccC51 operon, markedly lower than that of the E. coli genome, and the presence of direct repeats suggest the possibility of horizontal transfer of this gene cluster.