Iron sulfur (Fe-S) clusters are essential protein cofactors for most life forms. In human mitochondria, the core Fe-S biosynthetic enzymatic complex (called SDUF) consists of NFS1, ISD11, ISCU2, and frataxin (FXN) protein components. Few mechanistic details are known for how this complex synthesizes Fe-S clusters and how these clusters are delivered to targets. Here circular dichroism and Mössbauer spectroscopies were used to reveal details of the Fe-S cluster assembly reaction on the SDUF complex. SDUF reactions generated [2Fe-2S] cluster intermediates that readily converted to stable [2Fe-2S] clusters bound to uncomplexed ISCU2.