Antimicrobial peptides (AMPs) are multifunctional compounds that may show antimicrobial and immunomodulatory activities. With the rapid increase in the incidence of multidrug-resistant bacteria, there is an enormous interest in AMPs as templates for the production of new antibiotics. However, there are concerns that the therapeutic administration of AMPs can select resistant strains. In order to distinguish between resistant and non-resistant strains and verify resistance specificity to AMPs, in this study a magainin I-resistant Escherichia coli model was used. First, the identity of all strains was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-MS, VITEK 2 and MicroScan, and the susceptible and magainin-resistant strains were successfully differentiated by MALDI-TOF-MS analysis. Furthermore, cross-resistances to a broad spectrum of antibiotics were evaluated, showing that all E. coli strains are susceptible to the drugs tested, suggesting that the resistance seems to be specific to AMPs. Finally, the specific resistance to magainin I compared with other AMPs was checked by microdilution. This experiment showed that the magainin MICs were 62 and 104 μM for susceptible and resistant strains, respectively. The other AMPs MICs were 3.4 μM to proline-arginine-rich 39-amino-acid peptide, 43 μM to porcine myeloid antimicrobial 23-amino-acid peptide-23 and 1.2 μM to cecropin P1 for all strains, demonstrating any additional resistance to peptides here evaluated, confirming that the resistance seems to be essentially specific to magainin I. In summary, the data reported here reinforce the proposal that magainin I seems not to be merely a membrane disruptor, probably showing additional molecular targets in pathogenic bacteria.The Journal of Antibiotics advance online publication, 7 May 2014; doi:10.1038/ja.2014.48.