Acinetobacter baumannii has successfully developed resistance against all common antibiotics, including colistin (polymyxin E), the last universally active drug against this pathogen. The possible widespread distribution of colistin-resistant A. baumannii strains may create an alarming clinical situation. In a previous work, we reported differences in lethal mechanisms between polymyxin B (PXB) and the cecropin A-melittin (CA-M) hybrid peptide CA(1-8)M(1-18) (KWKLFKKIGIGAVLKVLTTGLPALIS-NH2) on colistin-susceptible strains (J. M. Saugar, T. Alarcón, S. López-Hernández, M. López-Brea, D. Andreu, and L. Rivas, Antimicrob. Agents Chemother. 46:875-878, 2002). We now demonstrate that CA(1-8)M(1-18) and three short analogues, namely CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH2), its Nalpha-octanoyl derivative (Oct-KWKLFKKIGAVLKVL-NH2), and CA(1-7)M(5-9) (KWKLLKKIGAVLKVL-NH2) are active against two colistin-resistant clinical strains. In vitro, resistance to colistin sulfate was targeted to the outer membrane, as spheroplasts were equally lysed by a given peptide, regardless of their respective level of colistin resistance. The CA-M hybrids were more efficient than colistin in displacing lipopolysaccharide-bound dansyl-polymyxin B from colistin-resistant but not from colistin-susceptible strains. Similar improved performance of the CA-M hybrids in permeation of the inner membrane was observed, regardless of the resistance pattern of the strain. These results argue in favor of a possible use of CA-M peptides, and by extension other antimicrobial peptides with similar features, as alternative chemotherapy in colistin-resistant Acinetobacter infections.