The structure elucidation of azotobactin D, the fluorescent siderophore excreted by Azotobacter uinelandii strain D, has been accomplished by using essentially NMR techniques and FAB mass spectrometry. It is a chromopeptide possessing a chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline bound to a peptide chain of 10 amino acids constituted with D- and L-serine (2), L-homoserine (3), D-citrulline (1), D-N'-acetyl-Nb-hydroxyornithine (1), L-aspartic acid (1), D-threo-P-hydroxyaspartic acid (1), and glycine (1). The chromophore is located at the N-terminus of the peptide, and one of the homoserines is at its C-terminus. The latter lactonizes readily, yielding a high amount of azotobactin 6, which is in fact the major compound isolated after the purification steps. The chromophore has an S chiral center. The structure of this siderophore differs significantly from the structure proposed by Fukasawa et al. [Fukasawa, K., Goto, M., Sasaki, K., Hirata, Y., & Sato, S. (1972) Tetrahedron 28, 5359-5365] for the fluorescent peptide excreted similarly by A. uinelandii strain 0.