Fermenting cells were grown under Fe-deficient and Fe-overload conditions, and their Fe contents were examined using biophysical spectroscopies. The high-affinity Fe import pathway was active only in Fe-deficient cells. Such cells contained ~150 μM Fe, distributed primarily into nonheme high-spin (NHHS) Fe(II) species and mitochondrial Fe. Most NHHS Fe(II) was not located in mitochondria, and its function is unknown. Mitochondria isolated from Fe-deficient cells contained [Fe(4)S(4)](2+) clusters, low- and high-spin hemes, S = (1)/(2) [Fe(2)S(2)](+) clusters, NHHS Fe(II) species, and [Fe(2)S(2)](2+) clusters. The presence of [Fe(2)S(2)](2+) clusters was unprecedented; their presence in previous samples was obscured by the spectroscopic signature of Fe(III) nanoparticles, which are absent in Fe-deficient cells. Whether Fe-deficient cells were grown under fermenting or respirofermenting conditions had no effect on Fe content; such cells prioritized their use of Fe to essential forms devoid of nanoparticles and vacuolar Fe. The majority of Mn ions in wild-type yeast cells was electron paramagnetic resonance-active Mn(II) and not located in mitochondria or vacuoles. Fermenting cells grown on Fe-sufficient and Fe-overloaded medium contained 400-450 μM Fe. In these cells, the concentration of nonmitochondrial NHHS Fe(II) declined 3-fold, relative to that in Fe-deficient cells, whereas the concentration of vacuolar NHHS Fe(III) increased to a limiting cellular concentration of ~300 μM. Isolated mitochondria contained more NHHS Fe(II) ions and substantial amounts of Fe(III) nanoparticles. The Fe contents of cells grown with excessive Fe in the medium were similar over a 250-fold change in nutrient Fe levels. The ability to limit Fe import prevents cells from becoming overloaded with Fe.