tRNA mG10/mG10 methyltransferase (archaeal Trm11) methylates the 2-amino group in guanosine at position 10 in tRNA and forms ,-dimethylguanosine (mG10) via -methylguanosine (mG10). We determined the complete sequence of tRNA, one of the substrate tRNAs for archaeal Trm11 from , a hyperthermophilic archaeon. Liquid chromatography/mass spectrometry following enzymatic digestion of tRNA identified 15 types of modified nucleoside at 21 positions. Several modifications were found at novel positions in tRNA, including 2'--methylcytidine at position 6, 2-thiocytidine at position 17, 2'--methyluridine at position 20, 5,2'--dimethylcytidine at position 32, and 2'--methylguanosine at position 42. Furthermore, methylwyosine was found at position 37 in this tRNA, although 1-methylguanosine is generally found at this location in tRNA from other archaea. We constructed (Δ) and some gene disruptant strains and compared their tRNA with that of the wild-type strain, which confirmed the absence of mG10 and other corresponding modifications, respectively. The lack of 2-methylguanosine (mG) at position 67 in the double disruptant strain suggested that this methylation is mediated by Trm14, which was previously identified as an mG6 methyltransferase. The Δ strain grew poorly at 95°C, indicating that archaeal Trm11 is required for survival at high temperatures. The mG10 modification might have effects on stabilization of tRNA and/or correct folding of tRNA at the high temperatures. Collectively, these results provide new clues to the function of modifications and the substrate specificities of modification enzymes in archaeal tRNA, enabling us to propose a strategy for tRNA stabilization of this archaeon at high temperatures. is a hyperthermophilic archaeon that can grow at 60 to 100°C. The sequence of tRNA from this archaeon was determined by liquid chromatography/mass spectrometry. Fifteen types of modified nucleoside were observed at 21 positions, including 5 modifications at novel positions; in addition, methylwyosine at position 37 was newly observed in an archaeal tRNA The construction of (Δ) and other gene disruptant strains confirmed the enzymes responsible for modifications in this tRNA. The lack of 2-methylguanosine (mG) at position 67 in the double disruptant strain suggested that this position is methylated by Trm14, which was previously identified as an mG6 methyltransferase. The Δ strain grew poorly at 95°C, indicating that archaeal Trm11 is required for survival at high temperatures.