Microcin D93 is an antibiotic substance produced by Escherichia coli strains which harbor the 5.5-kilobase plasmid pMccD93. Its production is unaffected by the use of different carbon and ammonia sources, different phosphate concentrations, or mitomycin C. We developed a method for purifying this microcin based on gel permeation chromatography and reverse-phase high-pressure liquid chromatography. The antibiotic appears to be a small, hydrophilic, basic peptide, active on E. coli and Proteus, Citrobacter, and Pseudomonas species and much more active on recA strains than on their isogenic wild type. Diminution of the rate of DNA biosynthesis without any apparent effect on other macromolecules appears to be a primary effect in the action of microcin D93.