The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015-1022, 2010) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5-30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.