Antimicrobial peptides (AMPs) have received much attention as models for the development of antibiotics capable of meeting the challenge of drug-resistant infections. Our research has focused on a linear, 15-residue cecropin-mellitin (CM) hybrid peptide designated CM15. In these studies we compare the biological activities and membrane interactions of CM15 and lysine substituted derivates designed to have optimized amphipathicity upon membrane binding. Previous studies have shown that these lysine enriched peptides maintain the high antimicrobial activity of the parent peptide but have substantially decreased hemolytic effects (Sato and Feix, Antimicrob. Agents Chemother. 52, 4463, 2008). Using model membranes to understand the differences that govern peptide-membrane interaction, we have performed a series of biophysical experiments including circular dichroism (CD), fluorescence, and site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy. These experiments establish the structures of the membrane-bound peptides, determine the extent of membrane lysis, and allow determination of partition coefficients and depth of penetration. Our results show that differences in red cell hemolysis can be reconstructed using model membranes, and provide insights into the mechanism of membrane disruption. This work was supported by award number R01GM068829 from the National Institute Of General Medical Sciences.