An antifungal protein (AFP), holotricin 3, was purified from the hemolymph of the coleopteran insect Holotrichia diomphalia larvae. Analysis of its cDNA showed that holotricin 3 is a novel Gly- and His-rich protein consisting of 84 amino acid residues. This protein was similar to AFP, an antifungal protein of Sarcophaga peregrina that was reported previously, in terms of molecular size and high content of Gly and His residues. However, no appreciable sequence similarity was detected between the two proteins.