The emergence of multidrug-resistant microbes has serious implications for managing infection and sepsis and has stimulated efforts to develop alternative treatments, such as antimicrobial peptides. The objective of this study was to test a designer peptide, novispirin G10, against multidrug-resistant microorganisms. By two-stage radial diffusion assays, its activity against such organisms compared favorably with that of standard antibiotics and other antimicrobial peptides. It killed bacteria very rapidly, was nonhemolytic, and was relatively noncytotoxic. The peptide induced an immediate, massive efflux of potassium from Pseudomonas aeruginosa, suggesting that it altered the permeability of its inner membrane. The presence of human serum reduced but did not eliminate its activity. We tested the in vivo activity of novispirin G10 in rats with an infected, partial-thickness burn that covered 20% of their total body surface area. The burned area was seeded with 10(6) CFU of a Silvadene-resistant P. aeruginosa strain, and 24 h later a single treatment with 0, 1, 3, or 6 mg of synthetic novispirin G10 (n = 16 at each concentration) per kg was given intradermally. Significant bacterial killing (P < 0.0001) was evident within 4 h in each peptide group compared to controls receiving vehicle. Antimicrobial peptides such as novispirin G10 may provide a useful alternative or adjunct to standard antibiotic agents in treating burns or other wound infections.