We screened an endoparasitic wasp (Pteromalus puparum) cDNA library for DNA sequences having antimicrobial activity using a vital dye exclusion assay. Two dozens of clones were isolated that inhibited the growth of host Escherichia coli cells due to expression of the cloned genes. Three peptides (PP13, PP102 and PP113) were synthesized chemically based on the amino acid sequences deduced from these clones and assayed for their antimicrobial activity. These peptides have net positive charges and are active against both Gram-negative and -positive bacteria, but are not active against fungi tested. Their hemolytic activity on human red blood cells was measured, and no hemolytic activity was observed after 1-h incubation at a concentration of 62.5 microM or below. A Blast search indicated that the three peptides have not been previously characterized as antimicrobial peptides (AMPs). Salt-dependency studies revealed that the biocidal activity of these peptides against E. coli decreased with increasing concentration of NaCl. Transmission electron microscopic (TEM) examination of PP13-treated E. coli cells showed extensive damage of cell membranes. The CD spectroscopy studies noted that the enhanced alpha-helical characteristics of PP13 strongly contribute to its higher antimicrobial properties. These results demonstrate the feasibility to identify novel AMPs by screening the expressional cDNA library.