<jats:p>Two ferric ion‐binding compounds, designated staphyloferrin A and B, were detected in the culture filtrates of staphylococci‐grown under iron‐deficient conditions. Staphyloferrin A was isolated from cultures of<jats:italic>Staphylococcus hyicus</jats:italic>DSM 20459. The structural elucidation of this highly hydrophilic, acid‐labile compound revealed a novel siderophore,<jats:italic>N</jats:italic><jats:sup>2</jats:sup>,<jats:italic>N</jats:italic><jats:sup>5</jats:sup>‐di‐(l‐oxo‐3‐hydroxy‐3,4‐dicarboxybutyl)‐D‐ornithine, which consists of one ornithine and two citric acid residues linked by two amide bonds. The two citric acid components of staphyloferrin A provide two tridentate pendant ligands, comprising of a β‐hydroxy, β‐carboxy‐substituted carboxylic acid derivative, for octahedral metal chelation. The CD spectrum of the staphyloferrin A ferric complex indicates a predominant A configuration about the ferric ion center. The uptake of ferric staphyloferrin A by<jats:italic>S. hyicus</jats:italic>obeys Michaelis‐Menten kinetics (<jats:italic>K</jats:italic><jats:sub>m</jats:sub>= 0.246 μM;<jats:italic>v</jats:italic><jats:sub>max</jats:sub>= 82 pmol · mg<jats:sup>−1</jats:sup>· min<jats:sup>−1</jats:sup>), indicating active transport of this siderophore. The staphyloferrin A transport system is different from that of the ferrioxamines as shown by an antagonism test. Production of staphyloferrin A is strongly iron‐dependent and is stimulated by supplementation of the medium with either D‐ or L‐ornithine. DL‐[5‐<jats:sup>14</jats:sup>C]ornithine was incorporated into staphyloferrin A, demonstrating that ornithine is an intermediate in staphyloferrin A biosynthesis.