Technical Abstract: Studies have indicated that Fe travels in the phloem as a low molecular weight (LMW) molecule. Here, we have focused on the wild-type cultivar Sparkle, and its two Fe-hyperaccumulating mutants, dgl and brz. The dgl mutant previously has been shown to transport excess Fe via the phloem. We have characterized the LMW protein composition of pea phloem exudate using matrix-assisted laser desorption time of flight mass spectrometry, immobilized metal affinity chromatography, and SDS-PAGE analysis. We have identified a potential Fe-binding peptide from this exudate, but because of its low abundance we also have attempted to isolate it in higher amounts from pea seed coat tissues. For phloem exudate from wild-type pea and the dgl mutant, the Fe-binding peptide exhibited a mass of 2.8 kDa estimated by SDS-PAGE. However, in seed coat tissues extracted under non-denaturing conditions, Fe-binding peptides (from 59Fe-labeled plants) separated by gel lfiltration chromatography were revealed to have approximate masses of 14 kDa in the wild-type and brz mutant, but only 3.5 kDa in the case of the dgl mutant. The discrepancy between phloem and seed coat samples suggests that in situ, Fe is bound as part of a molecular complex in both phloem and seed coats of wild-type pea and the brz mutant, and that this complex dissociates under stringent extracting conditions. For the dgl mutant, the lower molecular mass of the Fe-binding peptide, even under non-denaturing conditions, suggests that this complex is not formed in the phloem, perhaps due to a mutation in the peptide. Amino acid compositional analysis of this peptide from wild-type pea revealed the presence of cysteine (2.4%) and glutamic acid (14.2%), two potential ligands for Fe.