Iron-sulfur (Fe-S) clusters are versatile biological cofactors that require biosynthetic systems in vivo to be assembled. In Escherichia coli, the Isc (iscRSUA-hscBA-fdx-iscX) and Suf (sufABCDSE) pathways fulfill this function. Despite extensive biochemical and genetic analysis of these two pathways, the physiological function of the A-type proteins of each pathway (IscA and SufA) is still unclear. Studies conducted in vitro suggest two possible functions for A-type proteins, as Fe-S scaffold/transfer proteins or as iron donors during cluster assembly. To resolve this issue, SufA was coexpressed in vivo with its cognate partner proteins from the suf operon, SufBCDSE. Native SufA purified anaerobically using this approach was unambiguously demonstrated to be a [2Fe-2S] protein by biochemical analysis and UV-vis, Mossbauer, resonance Raman, and EPR spectroscopy. Furthermore, native [2Fe-2S] SufA can transfer its Fe-S cluster to both [2Fe-2S] and [4Fe-4S] apoproteins. These results clearly show that A-type proteins form Fe-S clusters in vivo and are competent to function as Fe-S transfer proteins as purified. This study resolves the contradictory results from previous in vitro studies and demonstrates the critical importance of providing in vivo partner proteins during protein overexpression to allow correct biochemical maturation of metalloproteins.