The trmD gene, which governs the formation of 1-methyl-guanosine(m1G) in transfer ribonucleic acid (tRNA), has been located by phage P1 transduction at 56 min on the chromosomal map of Escherichia coli. Cotransduction to tyrA at 56 min is 80%. From the Clarke and Carbon collection a ColE1-tyrA+ hybrid plasmid was isolated, which carried the trmD+ gene and was shown to over-produce the tRNA(m1G)methyltransferase. By subcloning restriction enzyme fragments in vitro, the trmD+ gene was located to a 3.4 kb DNA fragment 6.5 kb clockwise from the tyrA+ gene. The mutation trmD1, which renders the tRNA(m1G)methyltransferase temperature-sensitive both in vivo and in vitro could be complemented by trmD+ plasmids. These results suggest that the gene trmD+ is the structural gene for the tRNA(m1G)methyltransferase (EC 2.1.1.3.1).