Of the five Escherichia coli mutants thus far studied which lack 4-thiouridine (s4U) in tRNA, only one lacks the second of the two thiolating enzymes. All five strains have an otherwise normal thionucleotide pattern, and all five developed a thiamin (thiazole) requirement simultaneously with the s4U- state. The thioguanine derivative produced after infection of E. coli with bacteriophage lambda is still unidentified. Further information was collected on the lambda genes involved in this thiolation system.