We report here the isolation of three isoforms of a novel C-terminally amidated peptide from the gills of red sea bream, Chrysophrys (Pagrus) major. Peptide sequences were determined by a combination of Edman degradation, MS and HPLC analysis of native and synthetic peptides. Three peptides, named chrysophsin-1, chrysophsin-2, and chrysophsin-3, consist of 25, 25, and 20 amino acids, respectively, and are highly cationic, containing an unusual C-terminal RRRH sequence. The a-helical structures of the three chrysophsin peptides were predicted from their secondary structures and were confirmed by CD spectroscopy. The synthetic peptides displayed broad-spectrum bactericidal activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Bacillus subtilis, and fish and crustacean pathogens. The three peptides were also hemolytic. Immunohistochemical analysis showed that chrysophsins were localized in certain epithelial cells lining the surface of secondary lamellae and eosinophilic granule cell-like cells at the base of the secondary lamellae in red sea bream gills. Their broad ranging bactericidal activities, combined with their localization in certain cells and eosinophilic granule cell-like cells in the gills, suggest that chrysophsins play a significant role in the innate defense system of red sea bream gills.Keywords: antimicrobial peptide; chrysophsin; gills; red sea bream; synthetic peptide.Antimicrobial peptides are widely distributed throughout the animal and plant kingdoms [1]. They display a broad spectrum of antimicrobial activity against bacteria, yeast and filamentous fungi, and are recognized as an essential component in the first line of the host defense system [2,3].There are three major sites by which bacteria enter fish: the gills, gastrointestinal tract and skin [4]. Therefore, antibacterial substances are thought to exist at these sites to prevent penetration of bacteria into the circulatory system. In fact, skin mucus, eggs and serum of fish contain a variety of nonspecific defense substances, such as lysozyme, complement, C-reactive protein, transferrin, lectin, and antimicrobial proteins [5][6][7][8][9][10]. Furthermore, antimicrobial peptides have been purified from fish skin mucus: pardaxin from the moses sole fish Pardachirus marmoratus [11], pleurocidin from the winter flounder Pleuronectes americanus [12] and parasin I from the catfish Parasilurus asotus [13], and the gene expression of pleurocidin-like antimicrobial peptides found in the skin and intestine of the winter flounder [14]. The antimicrobial peptide, misgurin, has also been purified from the whole body of the loach Misgurnus anguillicaudatus [15]. These antibacterial peptides show potent antimicrobial activity against Gram-negative and Grampositive bacteria and act as nonspecific defense substances in fish skin.Fish gills are constantly being flushed with water that may contain fish pathogens, but are covered with only a thin layer of protective mucus and are constructed of only a single layer of fragile...