Microcin 24 is an antimicrobial peptide produced by Escherichia coli with activity against Salmonella typhimurium. In this study microcin 24 was investigated for its potential use in food safety, resistance development, and structure-activity relationship following site-directed mutagenesis. An attempt was made to produce microcin 24 from lactic acid bacteria by substituting the natural, dedicated leader peptide microcin-producing gene with a signal leader from divergicin A, which accesses the general secretion pathway. RNA sequences could be recovered from Carnobacterium divergens harboring an expression vector with the fusion construct, but no microcin activity could be detected. Concurrently, development and mechanism of resistance to microcin 24 was investigated by repeatedly exposing susceptible S. typhimurium to the microcin. The exposed S. typhimurium harbored an inducible regulator, marR, of the multiple antibiotic resistance (mar) regulon. These experiments showed that S. typhimurium could become resistant to microcin 24 through activation of the mar system leading to concurrent resistance to ciprofloxacin, chloramphenicol, rifampin, and tetracycline. Subsequently, an experiment was performed to see if microcin 24-producing E. coli could reduce S. typhimurium shedding in pigs and if this exposure could lead to microcin 24 resistance and mar activation in vivo. The results did not provide evidence of an effect on Salmonella shedding or mar activation under the conditions of the experiment. Site-directed mutagenesis performed in a conserved region of microcin 24 indicated that lysine substitutions introduced into a proposed hydrophobic face of a predicted amphipathic alpha-helix eliminated or severely reduced microcin activity. Other mutations in the microcin 24 gene were shown to affect microcin activity as well. Additional experiments provided evidence that microcin 24 is a bifunctional molecule with membrane and endonuclease activity.