Anguibactin (1), a novel siderophore isolated from the fish pathogen Vibrio anguillarum 775 (pJM1), has been identified as ω-N-hydroxy-ω-N-[[2'-(2",3"-dihydroxyphenyl)thiazolin-4'-yl]carboxy]histamine. The structure determination was based on (i) single-crystal X-ray diffraction studies of its anhydro derivative (2), (ii) ¹H and ¹³C NMR spectroscopy, (iii) FAB mass spectrometry, and (iv) chemical degradation. Anhydroanguibactin (2) crystallizes in the monoclinic space group C2/c, a = 22.71 (5) Å, b = 8.052 (6) Å, c = 15.879 (18) Å, β = 95.72 (9)°, V = 2889.2 ų, and Z = 8. The structure has been determined from 2029 diffractometer data and refined to a final R = 0.048. Spectroscopic results have shown that anguibactin (1) differs from 2 by having (i) a thiazoline ring instead of a thiazole ring and (ii) a hydroxyl group on the peptide N atom of the histamine residue. Anguibactin belongs to a unique structural class of its own, with its backbone derived from ω-N-hydroxyhistamine, cysteine, and 2,3-dihydroxybenzoic acid. The structure of another related compound, copurified with anguibactin, was identified as 2-(2',3'-dihydroxyphenyl)thiazoline-4-carboxylic acid methyl ester (3). A novel siderophore (microbial iron transport compound), named anguibactin (1), has been isolated recently from the iron-deficient cultures of a fish pathogenic bacterium, Vibrio anguillarum 775. Preliminary investigations have revealed that anguibactin (1) is unique in several respects. The production and biological activity of anguibactin and the virulence of the bacteria are linked to the presence of a 65-kb plasmid, designated pJM1, in the organism. Anguibactin is a very important factor of virulence as demonstrated by its ability to cross-feed a siderophore-deficient receptor-proficient mutant of V. anguillarum, which allows the establishment of this organism in the host vertebrate. Anguibactin possesses an unusual molecular composition, C₁₅H₁₆N₄O₄S, and shows significant structural differences with all known classes of siderophores. Moreover, the anguibactin receptor system has been shown to be highly specific for anguibactin and inert toward a range of bacterial, fungal, and synthetic iron chelators. Attempts to reveal its complete structure have been hindered due to problems associated with its purification in sufficient quantity. However, some aspects of its structure, such as the presence of a catechol group, have already been established. We have achieved success in determining the chemical and molecular structure of one of its closely related derivatives, anhydroanguibactin (2), by single-crystal X-ray diffraction. On the basis of this structural information and on the results obtained with NMR and FAB mass spectrometry, we have determined the structure of anguibactin (1) and a related compound (3), which has been copurified from the culture medium. In this paper, we present the evidence for the structure of anguibactin.