IscS, a cysteine desulfurase implicated in the repair of Fe-S clusters, was recently shown to act as a sulfurtransferase in the biosynthesis of 4-thiouridine (s(4)U) in tRNA (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561-16568). In frame deletion of the iscS gene in Escherichia coli results in a mutant strain that lacks s(4)U in its tRNA. Assays of cell-free extracts isolated from the iscS(-) strain confirm the complete loss of tRNA sulfurtransferase activity. In addition to lacking s(4)U, the iscS(-) strain requires thiamin and nicotinic acid for growth in minimal media. The thiamin requirement can be relieved by the addition of the thiamin precursor 5-hydroxyethyl-4-methylthiazole, indicating that iscS is required specifically for thiazole biosynthesis. The growth rate of the iscS(-) strain is half that of the parent strain in rich medium. When the iscS(-) strain is switched from rich to minimal medium containing thiamin and nicotinate, growth is preceded by a considerable lag period relative to the parent strain. Addition of isoleucine results in a significant reduction in the duration of this lag phase. To examine the thiazole requirement, we have reconstituted the in vitro biosynthesis of ThiS thiocarboxylate, the ultimate sulfur donor in thiazole biosynthesis, and we show that IscS mobilizes sulfur for transfer to the C-terminal carboxylate of ThiS. ThiI, a known factor involved in both thiazole and s(4)U synthesis, stimulates this sulfur transfer step by 7-fold. Extracts from the iscS(-) strain show significantly reduced activity in the in vitro synthesis of ThiS thiocarboxylate. Transformation of the iscS(-) strain with an iscS expression plasmid complemented all of the observed phenotypic effects of the deletion mutant. Of the remaining two nifS-like genes in E. coli, neither can complement loss of iscS when each is overexpressed in the iscS(-) strain. Thus, IscS plays a significant and specific role at the top of a potentially broad sulfur transfer cascade that is required for the biosynthesis of thiamin, NAD, Fe-S clusters, and thionucleosides.