The ferric iron-binding protein (Fbp) functions as a periplasmic-binding protein in the high-affinity active transport of growth-essential iron by pathogenic Neisseria. Fbp reversibly binds a single ferric ion per molecule of protein with high affinity. Similarly, the transferrins are a highly conserved family of bilobed vertebrate proteins that reversibly bind a single molecule of iron on each of the N- and C-terminal lobes. While evolutionarily divergent, iron binding by all described transferrin lobes is accomplished by a remarkably similar repertoire of residues, including two Tyr, one His, and one Asp, as well as a synergestic bicarbonate anion. With a molecular mass of ca. 34 kDa, Fbp approximates the size of a transferrin lobe. Given the similarities in iron-binding properties, it was investigated whether Fbp bound iron by a similar molecular strategy as the transferrins. The studies reported here demonstrate that the spectral properties of purified Fbp and human transferrin are similar in the visible range. Chemical modification of purified Fbp in the presence and absence of iron using the Tyr-specific modifier tetranitromethane demonstrates that between two and three Tyr residues are implicated in iron binding. A similar experiment using the His-specific reagent diethyl pyrocarbonate indicates that one of the six Fbp-encoded His residues is protected by iron. In addition, like the transferrins, a bicarbonate anion is required for the efficient coordination of iron by Fbp. The range of metals bound by Fbp and human transferrin, including the luminescent lanthanide terbium, is identical. Finally, terbium derivatives of Fbp and human transferrin yield virtually identical luminescence excitation spectra, implying a highly similar binding site environment. These studies suggest that the prokaryotic Fbp is a mono-sited analog for iron binding by the eukaryotic transferrins.