AbstractOn‐line capillary liquid chromatography–electrospray mass spectrometry and tandem mass spectrometry, in combination with precursor feeding and acetylation studies, were used to identify and characterize the siderophores of the fire blight pathogen Erwinia amylovora. Proferrioxamines D2, E, G1, G2, X1, and X2 were found, with D2 and E being the major siderophores for all strains of E. amylovora studied. These proferrioxamines have previously been observed with other microbial species. In addition, eleven additional hydroxamate compounds were found, nine of which occur naturally and two which were induced by directed fermentation. The novel natural proferrioxamines included three cyclic tetrahydroxamates, designated T1–3. The most abundant of these, T1, consists of four 5‐succinylamino‐1‐hydroxyaminopentane residues. In T2 and T3, one of these residues is substituted by 4‐succinylamino‐1‐hydroxyaminobutane and 3‐succinylamino‐1‐hydroxyaminopropane, respectively. E. amylovora also incorporated 3‐succinylamino‐1‐hydroxyaminopropane into a novel trihydroxamate, which has been designated X7. The novel finding that proferrioxamines may partly be comprised of 1,3‐diaminopropane residues is noteworthy because previous feeding studies with Streptomyces olivaceus Tü 2718 suggested that 1,3‐diaminopropane is biosynthetically not tolerated. A truncated proferrioxamine G1 was also identified, which is referred to as G1t, and, on feeding of diaminobutane, tetrahydroxamates T7 and T8, which are derived from T1 by substitution of two and three diaminopentane with diaminobutane residues. The structures of five other hydroxamates, with molecular masses of 530 (X8), 544 (X9), 622 (T4), 719 (T5) and 733 (T6), are not yet known. The results prove that the biosynthetic capabilities of E. amylovora for proferrioxamine production are flexible. E. amylovora may therefore be a good organism for the fermentative production of novel proferrioxamines that may be useful for reversing or avoiding aluminum and iron intoxications in man, for tumor diagnosis and therapy with radiolabeled antibodies and for use as DNA‐cleaving reagents. On the other hand, the production of all the different proferrioxamines involves common biosynthetic steps, some of which are not used in higher plants or vertebrates. Interference with the biosynthesis of proferrioxamines may therefore provide an opportunity for the development of alternative fire blight control agents.