Abstract: Biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is an essential process involving the mitochondrial iron-sulfur cluster (ISC) assembly and export machineries and the cytosolic Fe/S protein assembly (CIA) apparatus. To define the integration of Fe/S protein biogenesis into cellular homeostasis, we compared the global transcriptional responses to defects in the three biogenesis systems in S. cerevisiae using DNA microarrays. Depletion of a member of the CIA machinery elicited only weak (up to twofold) alterations in gene expression with no clear preference for any specific cellular process. In contrast, depletion of components of the mitochondrial ISC assembly and export systems induced strong and largely overlapping transcriptional responses of more than 200 genes (2-100-fold changes). These alterations were strikingly similar, yet not identical, to the transcriptional profiles developed upon iron starvation. Hence, mitochondria and their ISC systems serve as primary physiological regulators exerting a global control of numerous iron-dependent processes. First, ISC depletion activates the iron-responsive transcription factors Aft1/2p leading to increased cellular iron acquisition. Second, respiration and heme metabolism is repressed assuring the balanced utilization of iron by the two major iron-consuming processes, iron-sulfur protein and heme biosynthesis. Third, the decreased respiratory activity is compensated by induction of genes involved in glucose acquisition. Finally, transcriptional remodelling of the citric acid cycle, the biosyntheses of ergosterol and biotin integrate the iron dependence of these pathways. Together, our data suggest a model in which mitochondria perform a global regulatory role in numerous cellular processes linked to iron homeostasis.