Iron–sulfur (Fe–S) clusters are cofactors found in many proteins that have important redox, catalytic or regulatory functions. In mammalian cells, almost all known Fe–S proteins are found in the mitochondria, but at least one is found in the cytosol. Here we report cloning of the human homologs to IscU and NifU, iron-binding proteins that play a critical role in Fe–S cluster assembly in bacteria. In human cells, alternative splicing of a common pre-mRNA results in synthesis of two proteins that differ at the N-terminus and localize either to the cytosol (IscU1) or to the mitochondria (IscU2). Biochemical analyses demonstrate that IscU proteins specifically associate with IscS, a cysteine desulfurase that is proposed to sequester inorganic sulfur for Fe–S cluster assembly. Protein complexes containing IscU and IscS can be found in the mitochondria as well as in the cytosol, implying that Fe–S cluster assembly takes place in multiple subcellular compartments in mammalian cells. The possible roles of the IscU proteins in mammalian cells and the potential implications of compartmentalization of Fe–S cluster assembly are discussed.