A novel homolog of insect defensin, designated lucifensin II (Lucilia cuprina Wiedemann [Diptera: Calliphoridae] defensin), was purified from hemolymph extract from larvae of the blowfly L. cuprina. The full-length primary sequence of this peptide of 40 amino acid residues and three intramolecular disulfide bridges was determined by electrospray ionization-orbitrap mass spectrometry and Edman degradation and is almost identical to the previously identified sequence of lucifensin (Lucilia sericata Meigen defensin). Lucifensin II sequence differs from that of lucifensin by only one amino acid residue, that is, by isoleucine instead of valine at position 11. The presence of lucifensin II also was detected in the extracts of other larval tissues, such as gut, salivary glands, fat body, and whole body extract.