We have studied how puroindoline-b (PINB) mutants bind to model eukaryotic membranes dependent on binary composition of anionic:zwitterionic phospholipids and the presence of cholesterol and sphingomyelin in the model membrane. We have found that the trends in lipid binding a 6 behavior are different for wild-type PINB compared to its naturally occurring PINB(Trp(44)Arg) mutant form and have seen evidence of protein-induced domain formation within the lipid layer structure. Results show that selective binding of antimicrobial peptides to different membrane types is as a result of differences in lipid composition and the arrangement of lipids within the membrane surface. However, membrane binding behavior is not easily predicted; it is determined by net charge, hydrophobicity, and the amphiphilicity of the protein/peptide lipid-binding domain.