Recent computation analyses have pointed out an existence of conserved amino acid motifs among RNA ribose 2'-O-methyltransferases. However, the functions of these motifs are unclear yet. We carried out the site-directed mutagenesis studies systematically on Thermus thermophilus HB8 tRNA (Gm18) methyltransferase gene. Subsequent biochemical analyses with purified variant enzymes clearly revealed that five conserved amino acid residues (Asn35, Arg41, Glu124, Ser150, and Asn152) are involved in S-adenosyl-L-methionine binding. This is for the first time reporting the function of the conserved motifs among RNA ribose 2'-O-methyltransferases.