Saccharomyces cerevisiae responds to iron deprivation by increased transcription of the iron regulon, including the high affinity cell-surface transport system encoded by FET3 and FTR1 . Here we demonstrate that transcription of these genes does not respond directly to cytosolic iron but rather to the mitochondrial utilization of iron for the synthesis of iron-sulfur (Fe-S) clusters. We took advantage of a mutant form of an iron-dependent enzyme in the sterol pathway (Erg25-2p) to assess cytosolic iron levels. We showed that disruption of mitochondrial Fe-S biosynthesis, which results in excessive mitochondrial iron accumulation, leads to transcription of the iron transport system independent of the cytosolic iron level. There is an inverse correlation between the activity of the mitochondrial Fe-S-containing enzyme aconitase and the induction of FET3 . Regulation of transcription by Fe-S biosynthesis represents a mechanism by which cellular iron acquisition is integrated with mitochondrial iron metabolism.