Significance Synthesis of iron-sulfur clusters (ISC) and their insertion into apoproteins in eukaryotes requires the conserved, essential mitochondrial ISC and cytosolic Fe/S protein assembly machineries. Genetic mutations in most of the 18 different human ISC genes cause neurological, metabolic, and hematological diseases with often lethal outcome. Here, we have biochemically reconstituted the least-understood part of mitochondrial iron-sulfur protein assembly: The synthesis and insertion of [4Fe-4S] clusters. Apart from the in vivo-identified ISC factors (i.e., the [2Fe-2S] cluster donor GLRX5 and ISCA1–ISCA2–IBA57), this reaction specifically requires reduced ferredoxin FDX2, but not FDX1, for reductive fusion of [2Fe-2S] 2+ into [4Fe-4S] 2+ clusters. In contrast, [2Fe-2S] cluster transfer from GLRX5 to [2Fe-2S] target proteins occurs rapidly without any additional ISC factor.