Iron-sulfur (Fe-S) clusters serve as cofactors in many proteins that have important redox, catalytic, and regulatory functions. In bacteria, biogenesis of Fe-S clusters is mediated by multiple gene products encoded by the isc and nif operons. In particular, genetic and biochemical studies suggest that IscU, Nfu, and IscA function as scaffold proteins for assembly and delivery of rudimentary Fe-S clusters to target proteins. Here we report the characterization of human Nfu. A combination of biochemical and spectroscopic techniques, including UV-visible absorption and 57Fe Mössbauer spectroscopies, have been used to investigate the ability of purified human Nfu to assemble Fe-S clusters. The results suggest that Nfu can assemble approximately one labile [4Fe-4S] cluster per two Nfu monomers, and support the proposal that Nfu is an alternative scaffold protein for assembly of clusters that are subsequently used for maturation of targeted Fe-S proteins. Analyses of genomic DNA, transcripts, and translation products indicate that alternative splicing of a common pre-mRNA results in synthesis of two Nfu isoforms with distinct subcellular localizations. Isoform I is localized in the mitochondria, whereas isoform II is present in the cytosol and the nucleus. These results, together with previous reports of subcellular distributions of isoforms of human IscS and IscU in mitochondria, cytosol, and nucleus suggest that the Fe-S cluster assembly machineries are compartmentalized in higher eukaryotes.