The iron-dependent regulator (IdeR) protein in Mycobacterium tuberculosis, and its better characterized homologue, the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae, are iron-dependent regulatory proteins that control gene expression in response to iron availability in bacteria. IdeR regulates several genes required for iron uptake and storage including those involved in the synthesis of transition metal chelators called siderophores that are linked to the M. tuberculosis virulence. In this study, the metal ion and binding affinities for IdeR binding to an fxbA operator duplex DNA were estimated using fluorescence assays. The Fe(2+), Co(2+), and Ni(2+) affinities of the two metal ion binding sites in IdeR that are involved in the activation of the regulator DNA binding process in vitro were independently estimated. Binding to the two metal ion binding sites is apparently cooperative and the two affinities differ significantly. Occupation of the first metal ion binding site causes dimerization of IdeR, and the metal ion affinity is about 4 microM for Ni(2+) and much less for Fe(2+) and Co(2+). Binding of the second metal ion fully activates IdeR for binding to the fxbA operator. The equilibrium metal ion dissociation constants for IdeR-fxbA operator binding are approximately 9 microM for Fe(2+), 13 microM for Ni(2+), and 23 microM for Co(2+). Interestingly, the natural IdeR cofactor, Fe(2+), shows high affinities toward both binding sites. These results provide insight into the possible roles for each metal binding site in IdeR activation.