Iron–sulfur (Fe–S) clusters are versatile protein cofactors that require numerous components for their synthesis and insertion into apoproteins. In eukaryotes, maturation of cytosolic and nuclear Fe–S proteins is accomplished by cooperation of the mitochondrial iron–sulfur cluster (ISC) assembly and export machineries, and the cytosolic iron–sulfur protein assembly (CIA) system. Currently, nine CIA proteins are known to specifically assist the two major steps of the biogenesis reaction. They are essential for cell viability and conserved from yeast to man. The essential character of this biosynthetic process is explained by the involvement of Fe–S proteins in central processes of life, e.g., protein translation and numerous steps of nuclear DNA metabolism such as DNA replication and repair. Malfunctioning of these latter Fe–S enzymes leads to genome instability, a hallmark of cancer. This review is focused on the maturation and biological function of cytosolic and nuclear Fe–S proteins, a topic of central interest for both basic and medical research. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. Guest Editors: Roland Lill, Joan Broderick, and Dennis Dean.