A novel defensin-like peptide was identified in the greater wax moth, Galleria mellonella. It was discovered in a haemocyte cDNA bank enriched with transcripts upregulated after immune challenge via subtractive hybridisation and suppressive PCR. The deduced amino acid sequence of the defensin-like peptide exhibits similarities to the antifungal peptides drosomycin from Drosophila melanogaster and heliomicin from Heliothis virescens. Therefore, it has been termed gallerimycin. Upregulation of gallerimycin after stimulation of the immune system by LPS-injection was demonstrated by quantitative real-time PCR. A full-size cDNA was cloned and overexpressed in Escherichia coli Origami cells in order to obtain a functional peptide with disulfide bridges. The recombinant peptide was active against the entomopathogenic fungus Metarhizium anisopliae, but not against yeast, gram-negative and gram-positive bacteria. Arch. Insect Biochem. Physiol. 53:125–133, 2003. © 2003 Wiley-Liss, Inc.