The gene trmA, responsible for the production of 5-methyluridine (ribothymidine) in transfer ribonucleic acid, has been located at 79 min on the chromosomal map of Escherichia coli K-12. In five-factor crosses the gene order was shown to be argH-trmA-rif-thiA-metA. The co-transduction frequency between argH and trmA was 65%. Furthermore, the trmA5 mutation was shown to be recessive, in agreement with the notion that the trmA gene is the structural gene for the transfer tibonucleic acid (5-methyluridine) methyltransferase.