From a strain of Rhizopus microsporus var. rhizopodiformis a novel siderophore, named rhizoferrin, was isolated by ion-exchange column chromatography, gel filtration and preparative HPLC. Hydrolysis with 6 M HC1 and subsequent gas chromatography/mass spectrometry (GC/MS) of the esterified/trifluoroacetylated derivatives indicated that citric acid and diaminobutane were the only constituents. From positive fastatom-bombardment (FAB) and ion-spray tandem mass spectrometry, a molecular mass of 436 Da and the assignment of several daughter ion fragments could be obtained, which indicated the presence of two citric acid residues and one diaminobutane residue. NMR studies finally confirmed N1,N4-bis(1-oxo-3-hydroxy - 3,4-dicarboxybutyl)-diaminobutane as the structure of rhizoferrin. The iron-binding property was demonstrated on chromeazurol S plates and its siderophore activity was confirmed by iron transport measurements in young mycelia of R. microsporus. While rhizoferrin and also ferrioxamines B and E proved to be effective siderophores, coprogen was a poor siderophore in this fungus.