We have improved the in vitro assay for 4-thiouridine (s(4)U) biosynthesis in Escherichia coli tRNA by substituting an unmodified tRNA transcript as substrate and including recombinant ThiI protein, a known factor required for s(4)U synthesis. Using this assay, we have purified an enzyme from wild-type E. coli that is able to provide sulfur for s(4)U synthesis in vitro. The purified protein has a molecular weight of 45 kDa and contains pyridoxal phosphate as a cofactor. This protein catalyzes sulfur transfer from cysteine to tRNA and is analogous to factor C previously reported (Lipsett, M. N. (1972) J. Biol. Chem. 247, 1458-1461). UV spectroscopy and HPLC analysis of thiolated tRNA and its digests confirm that the product of the in vitro reaction is s(4)U. N-Terminal sequence analysis of the purified protein identifies it as IscS, a recently characterized NifS-like cysteine desulfurase that mobilizes sulfur for the synthesis of [Fe-S] clusters. We have cloned and overexpressed iscS and show that the recombinant protein displayed tRNA sulfurtransferase activity equal to that of the native protein. We also show that, of the multiple proteins in E. coli with cysteine desulfurase activity as observed by native gel staining, only IscS is able to mobilize the sulfur for transfer to tRNA. Our identification of IscS as a tRNA sulfurtransferase provides support for this activity in vivo and further expands the role for NifS proteins as versatile sulfur-carrying enzymes.