Rhizobactin 1021, a novel siderophore from the nitrogen-fixing alfalfa symbiont Rhizobium meliloti 1021, was isolated and structurally characterized by a combination of chemical and spectroscopic techniques. The compound is a citrate derivative in which the distal carboxyl groups of the molecule are amide linked to two different side chains. One of these is 1 -amino-3-(N-hydroxy-N-acetylamino)propane, and the other is l-amino-3-(N-hydroxy-N-(E)-2 deceny1amino)propane. The structure of rhizobactin 1021 was determined as (E)-4- [ [3-(acetylhydroxyamino)propyl] amino]-2-hydroxy-2- [2- [ 3- [hydroxy( 1-oxo-2-decenyl)amino]propyl]amino]-2-oxoethyl]-4-oxobutanoic acid. Ferric rhizobactin 1021 exists in solution predominantly in the A configuration, in an apparent equilibrium between a monomeric and a dimeric species.