The IscS protein is a pyridoxal phosphate-containing cysteine desulfurase involved in iron-sulfur cluster biogenesis. In prokaryotes, IscS is also involved in various metabolic functions, including thio-modification of tRNA. By contrast, the eukaryotic ortholog of IscS (Nfs1) has thus far been shown to be functional only in mitochondrial iron-sulfur cluster biogenesis. We demonstrate here that yeast Nfs1p is also required for the post-transcriptional thio-modification of both mitochondrial (mt) and cytoplasmic (cy) tRNAs in vivo. Depletion of Nfs1p resulted in an immediate impairment of the 2-thio-modification of 5-carboxymethylaminomethyl-2-thiouridine at the wobble positions of mt-tRNA(UUU)(Lys) and mt-tRNA(UUG)(Gln). In addition, we observed a severe reduction in the 2-thio-modification of 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) of cy-tRNA(UUU)(Lys2) and cy-tRNA(UUC)(Glu3), although the effect was somewhat delayed compared with that seen in mt-tRNAs. Mass spectrometry analysis revealed an increase in 5-methoxycarbonylmethyluridine concomitant with a decrease in mcm(5)s(2)U in cy-tRNAs that were prepared from Nfs1p-depleted cells. These results suggest that Nfs1p is involved in the 2-thio-modification of both 5-carboxymethylaminomethyl-2-thiouridine in mt-tRNAs and mcm(5)s(2)U in cy-tRNAs.