Pleurocidin (Pie) is a peptide derived from the winter flounder. In our previous study, we reported the antifungal effect of Pie and its mode of action. To develop novel antifungal peptides useful as therapeutic agents, two analogs, with amino acid substitutions, were designed to decrease the net hydrophobicity by Arg (R) or Ser (S)-substitution at the hydrophobic face of Pie without changing the amphipathic structure. By substituting Ser, the hydrophobicity of the peptide (anal-S) was decreased, and by substituting Arg, though the hydrophobicity of the peptide (anal-R) was decreased, the cationicity of this peptide was increased. CD measurements showed the substitution of Arg or Ser decrease the alpha-helical conformation of analog peptides. Studies with analog peptides have shown decreases in hydrophobicity and alpha-helicity do not affect antifungal activity but decrease hemolytic activity. These results suggest that highly hydrophobic and alpha-helical natures are not desirable in the design of antimicrobial peptides. (c) 2008 Elsevier Inc. All rights reserved.