IscS from Escherichia coli is a cysteine desulfurase that has been shown to be involved in Fe-S cluster formation. The enzyme converts L-cysteine to L-alanine and sulfane sulfur (S(0)) in the form of a cysteine persulfide in its active site. Recently, we reported that IscS can donate sulfur for the in vitro biosynthesis of 4-thiouridine (s(4)U), a modified nucleotide in tRNA. In addition to IscS, s(4)U synthesis in E. coli also requires the thiamin biosynthetic enzyme ThiI, Mg-ATP, and L-cysteine as the sulfur donor. We now report evidence that the sulfane sulfur generated by IscS is transferred sequentially to ThiI and then to tRNA during the in vitro synthesis of s(4)U. Treatment of ThiI with 5-((2-iodoacetamido)ethyl)-1-aminonapthalene sulfonic acid (I-AEDANS) results in irreversible inhibition, suggesting the presence of a reactive cysteine that is required for binding and/or catalysis. Both ATP and tRNA can protect ThiI from I-AEDANS inhibition. Finally, using gel shift and protease protection assays, we show that ThiI binds to unmodified E. coli tRNA(Phe). Together, these results suggest that ThiI is a recipient of S(0) from IscS and catalyzes the ultimate sulfur transfer step in the biosynthesis of s(4)U.