In our previous study (Chen et al. J Biol Chem 2005, 280:12316-12329), we utilized an alpha-helical antimicrobial peptide V(681) as the framework to study the effects of peptide hydrophobicity, amphipathicity, and helicity on biologic activities where we obtained several V(681) analogs with dramatic improvement in peptide therapeutic indices against gram-negative and gram-positive bacteria. In the present study, the D-enantiomers of three peptides--V(681), V13A(D) and V13K(L) were synthesized to compare biophysical and biologic properties with their enantiomeric isomers. Each D-enantiomer was shown by circular dichroism spectroscopy to be a mirror image of the corresponding L-isomer in benign conditions and in the presence of 50% trifluoroethanol. L- and D-enantiomers exhibited equivalent antimicrobial activities against a diverse group of Pseudomonas aeruginosa clinical isolates, various gram-negative and gram-positive bacteria and a fungus. In addition, L- and D-enantiomeric peptides were equally active in their ability to lyse human red blood cells. The similar activity of L- and D-enantiomeric peptides on prokaryotic or eukaryotic cell membranes suggests that there are no chiral receptors and the cell membrane is the sole target for these peptides. Peptide D-V13K(D) showed significant improvements in the therapeutic indices compared with the parent peptide V(681) by 53-fold against P. aeruginosa strains, 80-fold against gram-negative bacteria, 69-fold against gram-positive bacteria, and 33-fold against Candida albicans. The excellent stability of D-enantiomers to trypsin digestion (no proteolysis by trypsin) compared with the rapid breakdown of the L-enantiomers highlights the advantage of the D-enantiomers and their potential as clinical therapeutics.