Azotobacter vinelandii grown in iron-limited medium containing 1 μM molybdate released the catecholate siderophores azotochelin and aminochelin, but these were replaced by another catecholate compound when the medium contained 1 mM molybdate. Full and partial acid hydrolysis and fast atom bombardment mass spectroscopy showed the new compound was the tricatecholate protochelin, a condensation product of azotochelin and aminochelin. Protochelin production was iron-repressible and it rapidly decolorized the Chrome Azurol-S assay. Protochelin promoted growth of the siderophore-deficient A. vinelandii strain P100 under iron-restricted conditions and facilitated 55Fe uptake into iron-limited cells, confirming its function as a siderophore for A. vinelandii.