Fur (ferric uptake regulator) is the master regulator of iron homeostasis in many bacteria, but how it responds specifically to Fe(II) in vivo is not clear. Biochemical analyses of Bacillus subtilis Fur (BsFur) reveal that in addition to Fe(II), both Zn(II) and Mn(II) allosterically activate BsFur–DNA binding. Dimeric BsFur co-purifies with site 1 structural Zn(II) (Fur2Zn2) and can bind four additional Zn(II) or Mn(II) ions per dimer. Metal ion binding at previously described site 3 occurs with highest affinity, but the Fur2Zn2:Me2 form has only a modest increase in DNA binding affinity (approximately sevenfold). Metallation of site 2 (Fur2Zn2:Me4) leads to a ∼ 150-fold further enhancement in DNA binding affinity. Fe(II) binding studies indicate that BsFur buffers the intracellular Fe(II) concentration at ∼ 1 μM. Both Mn(II) and Zn(II) are normally buffered at levels insufficient for metallation of BsFur site 2, thereby accounting for the lack of cross-talk observed in vivo. However, in a perR mutant, where the BsFur concentration is elevated, BsFur may now use Mn(II) as a co-repressor and inappropriately repress iron uptake. Since PerR repression of fur is enhanced by Mn(II), and antagonized by Fe(II), PerR may co-regulate Fe(II) homeostasis by modulating BsFur levels in response to the Mn(II)/Fe(II) ratio.