Cationic cell penetrating peptides (CPPs) offer a promising, non-invasive platform for delivering synthetic or biological molecules that are otherwise impermeable to cell membranes. While the exact mechanism is unknown, cationic peptides are thought to adsorb at the lipid membrane/water interface, disrupt headgroup interactions, and produce local defects within the membrane that allow therapeutic molecules to enter the cell. In this contribution, we will describe the interactions between a series of low molecular weight, water-soluble tripodal cationic peptides (< 1000 MW) and lipid vesicles composed of zwitterionic (DPPC) or mixed zwitterionic/anionic (DPPC/DPPS or DPPC/DPPG) lipid bilayers examined by DSC, fluorescence spectroscopy, and cryo-TEM. The peptide was synthesized as Arg-C_n-Arg-C_n-Lys, where C_n represents the alkyl linkage separating the cationic residues (n = 4 to 11). Our results indicate that, despite being net-neutral, the melting temperature and cooperativity of DPPC was reduced with increasing C_n and peptide concentration. Bilayer disruption was more pronounced when anionic lipid was added as the peptides were able to bind to the negatively charged headgroups, significantly affecting melting behavior and inducing domain formation. The size and stability of these domains was controlled by C_n, peptide concentration, and/or lipid composition. In all cases, bilayer disruption increased with C_n. Our results are consistent with in vitro studies using human breast carcinoma xenograft cells that showed no uptake of a fluorescently labeled C₄ analog, but appreciable uptake of a labeled C₁₁ analog.