1-Methylinosine was isolated from unfractionated tRNAs of Sulfolobus acidocaldarius and Halobacterium volcani. The structure of 1-methylinosine was characterized by ultraviolet absorption and mass spectral analysis. 1-Methylinosine found in these archaebacterial tRNAs was located at residue 57 in T psi C-loop, indicating that the presence of 1-methylinosine is characteristic of archaebacteria.