TrmI generates N-1-methyladenosine at position 58 (m(1)A58) in tRNA. The Thermus thermophilus tRNA(Phe) transcript was methylated efficiently by T. thermophilus TrmI, whereas the yeast tRNA(Phe) transcript was poorly methylated. Fourteen chimeric tRNA transcripts derived from these two tRNAs revealed that TrmI recognized the combination of aminoacyl stem, variable region, and T-loop. This was confirmed by 10 deletion tRNA variants: TrmI methylated transcripts containing the aminoacyl stem, variable region, and T-arm. The requirement for the T-stem itself was confirmed by disrupting the T-stem. Disrupting the interaction between T- and D-arms accelerated the methylation, suggesting that this disruption is included in part of the reaction. Experiments with 17 point mutant transcripts elucidated the positive sequence determinants C56, purine 57, A58, and U60. Replacing A58 with inosine and 2-aminopurine completely abrogated methylation, demonstrating that the 6-amino group in A58 is recognized by TrmI. T. thermophilus tRNA(GGU)(GGU)(Thr)(Thr) contains C60 instead of U60. The tRNA(GGU)(Thr) transcript was poorly methylated by TrmI, and replacing C60 with U increased the methylation, consistent with the point mutation experiments. A gel shift assay revealed that tRNA(GGU)(Thr) had a low affinity for TrmI than tRNA(Phe). Furthermore, analysis of tRNA(GGU)(Thr) purified from the trmI gene disruptant strain revealed that the other modifications in tRNA accelerated the formation of m1A58 by TrmI. Moreover, nucleoside analysis of tRNA(GGU)(Thr) from the wild-type strain indicated that less than 50% of tRNA(GGU)(Thr) contained m(1)A58. Thus, the results from the in vitro experiments were confirmed by the in vivo methylation patterns.