Cysteine desulfurases generate a covalent persulfide intermediate from cysteine, and this activated form of sulfur is essential for the synthesis of iron–sulfur (Fe–S) clusters. In yeast mitochondria, there is a complete machinery for Fe–S cluster synthesis, including a cysteine desulfurase, Nfs1p. Here we show that following supplementation of isolated mitochondria with [ 35 S]cysteine, a radiolabeled persulfide could be detected on Nfs1p. The persulfide persisted under conditions that did not permit Fe–S cluster formation, such as nucleotide and/or iron depletion of mitochondria. By contrast, under permissive conditions, the radiolabeled Nfs1p persulfide was greatly reduced and radiolabeled aconitase was formed, indicating transfer of persulfide to downstream Fe–S cluster recipients. Nfs1p in mitochondria was found to be relatively more resistant to inactivation by N-ethylmaleimide (NEM) as compared with a prokaryotic cysteine desulfurase. Mitochondria treated with NEM (1mM) formed the persulfide on Nfs1p but failed to generate Fe–S clusters on aconitase, likely due to inactivation of downstream recipient(s) of the Nfs1p persulfide. Thus the Nfs1p-bound persulfide as described here represents a precursor en route to Fe–S cluster synthesis in mitochondria.