Recently two -defensins, named spheniscins, have been isolated from the stomach content of the king penguin (Aptenodytes patagonicus), which is capable of preserving food for several weeks during egg incubation (Thouzeau, C., Le Maho, Y., Froget, G., Sabatier, L., Le Bohec, C., Hoffmann, J. A., and Bulet, P. (2003) J. Biol. Chem. 278, 51053-51058). It has been proposed that, in combination with other antimicrobial peptides, spheniscins may be involved in this long term preservation of food in the bird's stomach. To draw some structure/ function features, the three-dimensional structure in aqueous solution of the most abundant spheniscin (Sphe-2) was determined by two-dimensional NMR and molecular modeling techniques. The overall fold of Sphe-2 includes a three-stranded antiparallel -sheet stabilized by three disulfide bridges with a pairing typical of -defensins. In addition, the N-terminal segment shows helical features on most structures. Sphe-2 is highly cationic, and its surface displays a hydrophobic patch. Comparative modeling revealed that this patch is preserved in avian defensins. The activity of Sphe-2 against a pathogenic Gram-positive strain was retained in vitro in the conditions of osmolarity found in penguin stomach content and also in different salt concentrations and compositions up to those reported for seawater. Comparison with structurally related mammalian -defensins showed that the hydrophobic patch is not preserved in mammalian -defensins and that the high cationicity of Sphe-2 is presumably the critical factor for its retained activity in high salt concentrations. Such peculiarities, in addition to a broad activity spectrum, suggest that penguin defensins may represent interesting probes for the design of highly efficient antibiotics to fight off pathogens that develop in relatively salt-rich body fluids.During the final stage of egg incubation in king penguins (Aptenodytes patagonicus), the male can preserve undigested food in the stomach for several weeks (1). This ensures survival of the newly hatched chick in the event that the return of the foraging female from the sea is delayed. In accordance with the characterization of stress-induced bacteria (2), a previous study has demonstrated that numerous antimicrobial activities exist in preserved stomach contents (3). Two antimicrobial peptides have been isolated and fully characterized, namely spheniscin-1 and -2 (Sphe-1/pBD-1 and Sphe-2/pBD-2).1 The two forms of spheniscins differ by a single residue, His 14 , in Sphe-1 versus Arg 14 in Sphe-2. A data bank search revealed that spheniscins are members of the well known defensin family.Defensins are small (3-5 kDa) cationic antimicrobial peptides that are part of the innate immunity of vertebrates (4, 5), invertebrates (6), and plants (7). In vertebrates, defensins can be divided into ␣-, -, and -defensin subfamilies. The unusual -defensin, which is a circular peptide, was initially isolated from rhesus macaque (8). Recently a pseudogene coding for a homologue, named retrocyclin, wa...