OBJECTIVES: Commercially produced sterile green bottle fly Lucilia sericata maggots are successfully employed by practitioners worldwide to clean a multitude of chronic necrotic wounds and reduce wound bacterial burdens during maggot debridement therapy (MDT). Secretions from the maggots exhibit antimicrobial activity along with other activities beneficial for wound healing. With the rise of multidrug-resistant bacteria, new approaches to identifying the active compounds responsible for the antimicrobial activity within this treatment are imperative. Therefore, the aim of this study was to use a novel approach to investigate the output of secreted proteins from the maggots under conditions mimicking clinical treatments. METHODS: cDNA libraries constructed from microdissected salivary glands and whole maggots, respectively, were treated with transposon-assisted signal trapping (TAST), a technique selecting for the identification of secreted proteins. Several putative secreted components of insect immunity were identified, including a defensin named lucifensin, which was produced recombinantly as a Trx-fusion protein in Escherichia coli, purified using immobilized metal affinity chromatography and reverse-phase HPLC, and tested in vitro against Gram-positive and Gram-negative bacterial strains. RESULTS: Lucifensin was active against Staphylococcus carnosus, Streptococcus pyogenes and Streptococcus pneumoniae (MIC 2 mg/L), as well as Staphylococcus aureus (MIC 16 mg/L). The peptide did not show antimicrobial activity towards Gram-negative bacteria. The MIC of lucifensin for the methicillin-resistant S. aureus and glycopeptide-intermediate S. aureus isolates tested ranged from 8 to >128 mg/L. CONCLUSIONS: The TAST results did not reveal any highly secreted compounds with putative antimicrobial activity, implying an alternative antimicrobial activity of MDT. Lucifensin showed antimicrobial activities comparable to other defensins and could have potential as a future drug candidate scaffold, for redesign for other applications besides the topical treatment of infected wounds.