Among hundreds of heterotrophic bacteria isolated from sediments of a lagoon near Lake Biwa, Japan, Alcaligenes denitrificans subsp. xylosoxydans KN 3-1 (positive for hydroxamate siderophore bioassay) was selected. The bacterium excreted a siderophore (designated alcaligin) into TTG medium after mid-logarithmic growth, with a final yield of ca. 1.3 mM in 10-day-old culture fluid. Isolation/purification involved Dowex 1x4 column, ammonium sulfate saturation extraction, and BioGel P-2 chromatography. FABMS and elemental analysis indicated the empirical formula C16H28N4O8·2H2O. Spectroscopic (UV, IR, 1H/13C NMR) data suggested two hydroxamate groups. Single-crystal X-ray crystallography revealed alcaligin's novel ring dihydroxamate structure. Job's plots showed it chelates ferric iron at molar ratios of 3:2 (pH 6.0) and 1:1 (pH 2.0). Apparent stability constants (EDTA displacement method) were smaller than those of EDTA or known dihydroxamate siderophores.