Background: Due to their chemical definition and reduced size, the use of peptides as gene delivery systems is gaining interest as compared to the more common polymeric non‐viral vectors. To achieve gene transfer efficiencies that would make peptides a realistic alternative to existing methods, we have evaluated and attempted to concert those properties with a direct impact on the activity of the system. These considerations have led to the design, synthesis and characterization of a 23‐residue cationic peptide which we term RAWA. Methods: We have characterized RAWA biophysically and functionally. Biophysical studies include evaluation of DNA condensation and membrane perturbing activities. DNA transfer activity has been evaluated in cell culture at controlled DNA‐to‐peptide stoichiometries, using a luciferase gene as reporter. Requirements for additional effectors such as chloroquine and peptide cofactors have also been considered. Results: RAWA displays in vitro DNA condensing activity similar to that of protamines, reaching maximum effect at a peptide‐to‐DNA molar charge ratio (CR) of 4 (+/−). The reduced membrane perturbing activity diminishes its cytotoxic potential. In COS‐7 cells, transfection efficiency with RAWA peptiplexes, compares favorably with well‐recognized systems, including Lipofectamine Plus, Superfect, GenePorter and FuGene. The peptide‐associated activity between free and DNA‐bound species has been mapped by analyzing dependency on chloroquine treatment. The lack of significant serum inhibition and low toxicity make this system advantageous for potential in vivo application. A ternary complex including the acid‐triggered fusogenic JTS‐1 peptide is presented as a potential strategy for further in vivo studies. Conclusions: We have developed a gene delivery system based on an amphipathic cationic peptide with improved DNA condensation ability and reduced cytotoxicity, which maintains membrane binding and perturbing activities. Observed efficiency with this molecule is very high and compares favorably with currently available transfection systems. Copyright © 2000 John Wiley & Sons, Ltd.