Recently, we reported on a new group of diastereomers of short-model peptides (12 amino acids long) composed of leucine and lysine with varying ratios, possessing several properties that make them potentially better than native or de novo-designed all L-amino acid antimicrobial peptides. Preliminary studies have revealed that modulating the hydrophobicity and positive charges of these diastereomers is sufficient to confer antibacterial activity and cell selectivity. However, the relationship between their biological function, structure, and mode of action was not investigated. Here we synthesized and investigated three types of linear model diastereomers (12 amino acids long) with varying lysine:leucine (or tryptophan) ratios (i.e., K(3)L(8)W, K(5)L(6)W, and K(7)L(4)W), which confer different levels of lytic activities. For each K:L ratio, tryptophan was introduced in the middle or the N- or C-terminus of the peptides, as an intrinsic fluorescent probe. Only the hemolytic peptide K(3)L(8)W binds to both negatively charged and zwitterionic phospholipid membranes. K(5)L(6)W and K(7)L(4)W bind similarly, but only to negatively charged membranes, despite the fact that K(5)L(6)W is substantially more lytic to bacteria than K(7)L(4)W. Interestingly, although K(3)L(8)W contains 33% D-amino acids, ATR-FTIR spectroscopy revealed a structure of approximately 90% alpha-helix in both types of membranes. In addition, K(5)L(6)W contains approximately 40% 3(10)-helix and K(7)L(4)W is predominantly a random coil in membranes. Polarized ATR-FTIR and tryptophan-quenching experiments, using brominated phospholipids, revealed a similar depth of penetration and an orientation that was parallel to the membrane surface for all the peptides, but with K(3)L(8)W affecting the lipid order more than the others. The results provide insight into the mode of action of this group of diastereomeric peptides, and the effect of hydrophobicity and positive charges on their membrane structure, function, and cell selectivity. Moreover, this research should assist in the development of suitable diastereomeric peptide antibiotics for therapeutic use that would overcome the problem the increasing resistance of bacteria to conventional antibiotics.