Many naturally occurring antimicrobial peptides comprise cationic linear sequences with the potential to adopt an amphipathic alpha-helical conformation. We designed a linear 18-residue peptide that adopted an amphipathic beta-sheet structure when it was bound to lipids. In comparison to a 21-residue amphipathic alpha-helical peptide of equal charge and hydrophobicity, this peptide possessed more similar antimicrobial activity and greater selectivity in binding to and inducing leakage in vesicles composed of bacterial membrane lipids than vesicles composed of mammalian membrane lipids (J. Blazyk, R. Weigand, J. Klein, J. Hammer, R. M. Epand, R. F. Epand, W. L. Maloy, and U. P. Kari, J. Biol. Chem. 276:27899-27906, 2001). Here, we compare two systematically designed families of linear cationic peptides to evaluate the importance of amphipathicity for determination of antimicrobial activity. Each peptide contains six lysine residues and is amidated at the carboxyl terminus. The first family consists of five peptides with various capacities to form amphipathic beta-sheet structures. The second family consists of six peptides with various potentials to form amphipathic alpha helices. Only those peptides that can form a highly amphipathic structure (either a beta sheet or an alpha helix) possessed significant antimicrobial activities. Striking differences in the abilities to bind to and induce leakage in membranes and lipid vesicles were observed for the two families. Overall, the amphipathic beta-sheet peptides are less lytic than their amphipathic alpha-helical counterparts, particularly toward membranes containing phosphatidylcholine, a lipid commonly found in mammalian plasma membranes. Thus, it appears that antimicrobial peptides that can form an amphipathic beta-sheet conformation may offer a selective advantage in targeting bacterial cells.