Iron-sulfur (Fe-S) proteins are localized in the cytosol, nucleus, and mitochondria of mammalian cells (reviewed in Stemmler et al. 2010, Rouault 2012, Bandyopadhyay et al. 2008, Lill 2009, Lill et al. 2012). Fe-S protein biogenesis in the mitochondrial matrix involves the iron-sulfur cluster (ISC) assembly machinery. Ferrous iron is transported across the inner mitochondrial membrane into the mitochondrial matrix by Mitoferrin-1 (SLC25A37) and Mitoferrin-2 (SLC25A28). (Mitoferrin-1 is enriched in erythroid cells while Mitoferrin-2 is ubiquitous.) Frataxin binds ferrous iron in the mitochondrial matrix. The cysteine desulfurase NFS1 in a subcomplex with ISD11 provides the sulfur by converting cyteine into alanine and forming a persulfide which is used for cluster formation on ISCU, the scaffold protein. Interaction between NFS1 and ISD11 is necessary for desulfurase activity. Frataxin binds to a complex containing NFS1, ISD11, and ISCU and is proposed to function as an iron donor to ISCU or as an allosteric switch that activates sulfur transfer and Fe-S cluster assembly (Tsai and Barondeau 2010). Cluster formation also involves the electron transfer chain ferredoxin reductase and ferredoxin. ISCU initially forms clusters containing 2 iron atoms and 2 sulfur atoms ([2Fe-2S] clusters). They are released by the function of HSP70-HSC20 chaperones and the monothiol glutaredoxin GLRX5 and used for assembly of [2Fe-2S] proteins. Assembly of larger clusters such as [4Fe-4S] clusters may involve the function of ISCA1, ISCA2, and IBA57. The clusters are transferred to apo-enzymes such as the respiratory complexes, aconitase, and lipoate synthase through dedicated targeting factors such as IND1, NFU1, and BOLA3.