Mitochondria are the site of assembly of FeS centers of mitochondrial and cytosolic FeS proteins. Various microaerophilic or anaerobic unicellular eukaryotes lack typical mitochondria ("amitochondriate" protists). In some of these organisms, a metabolically different organelle, the hydrogenosome, is found, which is thought to derive from the same proteobacterial ancestor as mitochondria. Here, we show that hydrogenosomes of Trichomonas vaginalis, a human genitourinary parasite, contain a key enzyme of FeS center biosynthesis, cysteine desulfurase (TviscS-2), which is phylogenetically related to its mitochondrial homologs. Hydrogenosomes catalyze the enzymatic assembly and insertion of FeS centers into apoproteins, as shown by the reconstruction of the apoform of [2Fe-2S]ferredoxin and the incorporation of 35S from labeled cysteine. Our results indicate that the biosynthesis of FeS proteins is performed by a homologous system in mitochondriate and amitochondriate eukaryotes and that this process is inherited from the proteobacterial ancestor of mitochondria.