The designer proline-rich antimicrobial peptide A3-APO and its Chex1-Arg20 single chain in vivo metabolite were studied for their ability to induce bacterial resistance upon repeated incubation of Escherichia coli and Klebsiella pneumoniae strains in sublethal concentrations. While no resistant E. coli phenotype emerged to either peptides, after 10 passages the K. pneumoniae strain became resistant to the monomer but not the dimer. The major microbiological difference between A3-APO and Chex1-Arg20 is the improved membrane-disintegrating ability of the dimeric prodrug. Thus, in agreement with earlier studies, the induced resistance likely resides in some membrane component rather than the intracellular target protein DnaK. In support, no genetic alteration in the DnaK multihelical lid region could be observed in any of the sensitive or resistant bacterial strains.