The increase in infectious diseases and bacterial resistance to antibiotics has resulted in intensive studies focusing on the use of linear, α-helical, cytolytic peptides from insects and mammals as potential drugs for new target sites in bacteria. Recent studies with diastereomers of the highly potent cytolytic peptides, pardaxin and melittin, indicate that α- helical structure is required for mammalian cells lysis but is not necessary for antibacterial activity. Thus, hydrophobicity and net positive charge of the polypeptide might confer selective antibacterial lytic activity. To test this hypothesis, a series of diastereomeric model peptides (12 amino acids long) composed of varying ratios of leucine and lysine were synthesized, and their structure and biological function were investigated. Peptide length and the position of D-amino acids were such that short peptides with stretches of only 1-3 consecutive L-amino acids that cannot form an α-helical structure were constructed. Circular dichroism spectroscopy showed that the peptides do not retain any detectable secondary structure in a hydrophobic environment. This enabled examination of the sole effect of hydrophobicity and positive charge on activity. The data reveal that modulating hydrophobicity and positive charge is sufficient to confer antibacterial activity and cell selectivity. A highly hydrophobic diastereomer that permeated both zwitterionic and negatively charged phospholipid vesicles, lysed eukaryotic and prokaryotic cells. In contrast, a highly positively charged diastereomer that only permeated slightly negatively charged phospholipid vesicles had low antibacterial activity and could not lyse eukaryotic cells. In the boundary between high hydrophobicity and high positive charge, the diastereomers acquired selective and potent antibacterial activity. Furthermore, they were completely resistant to human serum inactivation, which dramatically reduces the activity of native antibacterial peptides. In addition, a strong synergistic effect was observed at nonlethal concentrations of the peptides with the antibiotic tetracycline on resistant bacteria. The results are discussed in terms of proposed mechanisms of antibacterial activity, as well as a new strategy for the design of a repertoire of short, simple, and easily manipulated antibacterial peptides as potential drugs in the treatment of infectious diseases.