Since the position of the genes specifying the two enzymes involved in the formation of 4-thiouridine in E. coli tRNA is roughly known, a search is being made for E. coli mutants lacking one or the other of the enzymes. The first enzyme in the system is obtainable in homogeneous form, and will be used to investigate the structural requirements in the acceptor tRNA and the nature of the intermediate produced by the enzyme.