Microcin C7, a peptide antibiotic inhibitor of protein synthesis, is produced by Escherichia coli K-12 strains that carry the 43-kilobase low-copy-number plasmid pMccC7. Microcin C7 production and immunity determinants of this plasmid have been cloned into the vectors pBR322 and pACYC184. The resulting plasmids overproduce microcin C7 and express immunity against the microcin. Mcc- and Mcc- Imm- mutants have been isolated on recombinant plasmids by inserting transposable elements. Physical and phenotypic characterization of these mutants shows that a DNA region of 5 kilobases is required to produce microcin C7, and that two small regions located inside the producing region are also required to express immunity. Analysis of plasmids carrying mcc-lacZ gene fusions indicates that all microcin DNA is transcribed in the same direction. The results suggest that a structure like a polycistronic operon is responsible for microcin C7 production and immunity.