ABSTRACT Derivatives of the cytotoxic peptide dermaseptin S4 have recently emerged as potential antimicrobial agents. Here, we report on the antibacterial properties of three derivatives with improved toxicity profiles: a 28-residues K ₄ K ₂₀ -S4 and two shorter versions, K ₄ -S4(1-16) and K ₄ -S4(1-13). The range of MICs of K ₄ K ₂₀ -S4 against clinical isolates of Staphylococcus aureus , Pseudomonas aeruginosa, and Escherichia coli were, respectively, 1 to 4, 1 to 4, and 1 to 16 μg/ml. MICs of the short derivatives were rather similar or two to fourfold higher. Each of the three peptides was rapidly bactericidal in vitro, reducing the number of viable CFU of either E. coli or S. aureus by 6 log units in 30 min or less. Compared with MSI-78 or PG-1, K ₄ -S4(1-13) was at least as potent against bacteria (assessed at two MIC multiples) but displayed lesser toxicity against human erythrocytes. Serial passage in subinhibitory concentrations led to emergence of resistance to commercial antibiotics but not to the l - or d isomer of either of the dermaseptin derivatives. The short derivatives were further investigated for antibacterial activity in vivo, using a peritonitis model of mice infected with P. aeruginosa . Naive mice in the vehicle control group exhibited 75% mortality, compared to 18 or 36% mortality in mice that received a single intraperitoneal injection (4.5 mg/kg) of K ₄ -S4(1-16) or K ₄ -S4(1-13), respectively. In vivo bactericidal activity was confirmed in neutropenic mice, where intraperitoneal administration of K ₄ -S4(1-16) reduced the number of viable CFU in a dose-dependent manner by >3 log units within 1 h of exposure, and this was sustained for at least 5 h. Overall, the data suggest that dermaseptin S4 derivatives could be useful in treatment of infections, including infections caused by multidrug-resistant bacteria.