Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safety, stability, andsuitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factorin application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles toovercome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series ofnovel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysineresidues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA bindingat neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidineresidues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies.The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptidecomplex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexesformed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particlesizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion ofhistidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary forhigh transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene deliveryof the luciferase encoding plasmid, pCMV-Luc, mediated by all the lipopeptides, was much higher than poly(Llysine)(PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branchedpolyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidineresidue producedspontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved byincorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purifiedand offer new insights into the mechanism of action of transfection complexes.