The ferric uptake regulator is a metal-dependent transcription repressor that is activated by divalent transition metal cations. Fe(II) is believed to be the primary functional metal in vivo; however, the ability of other divalent cations to activate Fur brings into question the true physiological metal. Furthermore, the role of different oxidation states of iron in activating Fur has not been determined. Comparison of the affinity of different metals with intracellular metal concentrations would suggest which metals activate Fur in vivo; however, no accurate determinations of the affinity of Fur for metals have been reported. In this study, methods for reconstituting Fur with Fe(II), Fe(III), Co(II), and Zn(II) are described. Reconstituted protein was assayed for DNA affinity by gel shift assays. Fur is activated for DNA binding when reconstituted with Fe(III), as well as Fe(II), Zn(II), Co(II), and Mn(II), with little difference in DNA affinity for the different metallo forms of Fur. The affinity of Fur for the different metals was determined and ranges over several orders of magnitude in the following order: Zn(II) > Co(II) > Fe(II) > Mn(II). Only Fe(II) binds with sufficient affinity to activate Fur significantly at physiological metal concentrations, when compared to previously determined total metal concentrations in Escherichia coli.