Transfer RNA (guanosine-2'-)-methyltransferase (Gm-methylase, EC.2.1.1.32) from extreme thermophile, Thermus thermophilus HB27 is one of the tRNA-ribose modification enzymes; this enzyme specifically catalyze the transfer of a methyl group from S-adenosyl-L-methionine to 2'-OH of the ribose of the guanosine at position 18 in tRNA. A broad substrate specificity of Gm-methylase was observed using natural tRNAs as methyl group acceptors, which suggests that some local stractures common in tRNAs are recognized by the enzyme. By using yeast tRNA(Phe) variants obtained by transcription of their genes with T7 RNA polymerase, it was revealed that the residues G18 and G19, as well as the D-stem structure were primarily required for the methylation reaction and that the essentially minimal sequence for the substrate was Pyrimidine17-G18-G19. The other conserved sequences and the tertiary base-pairs were not essential, but G15, G46, U55 and C56 strongly affected the methylation efficiency.