The structure of vibrioferrin, a siderophore from Vibrio parahaemolyticus, was elucidated based on a combination of partial hydrolysis and spectroscopic techniques. HPLC of purified vibrioferrin showed two peaks with an area ratio of approximately 2:1. However, upon reinjection of each of those isolated compounds, the original chromatographic pattern was obtained, indicating an equilibrium between two compounds in aqueous solution. Consistent with this finding, most of the NMR signals of vibrioferrin were duplicated. The structure was determined as 1-(2-[2-(5-carboxy-5-hydroxy-2-oxo-1-pyrrolidinyl)propionamide]ethyl) citrate, which exists in two epimeric forms resulting from cyclization between an amidic nitrogen of the alanine residue and a keto group of the 2-ketoglutaric acid residue. Transport experiments with 55Fe-labeled vibrioferrin demonstrated the function of vibrioferrin as a siderophore in V. parahaemolyticus. Kinetic studies with mid-log phase cells revealed that the iron uptake system was receptor-mediated, with Km and Vmax values of 67 nM and 54 pmol Fe/mg cell protein/min, respectively. Moreover, iron uptake mediated by vibrioferrin was blocked both by uncouplers and by ATPase inhibitors.