A number of post-transcriptional modifications in tRNA are phylogenetically characteristic of the bacterial, eukaryal, or archaeal domains, both with respect to sequence location and molecular structure at the nucleoside level. One of the most distinct such modifications is nucleoside G*, located in archaeal tRNA at position 15, which in bacterial and eukaryal tRNAs is a conserved site involved in maintenance of the dihydrouridine loop-T-loop tertiary interactions. G* occurs widely in nearly every branch of the archaeal phylogenetic domain, in contrast to its absence in all reported bacterial and eukaryal tRNA sequences. The structure of G*-15 is 2-amino-4,7-dihydro-4-oxo-7-beta-D-ribofuranosyl-1H- pyrrolo[2,3-d]pyrimidine-5-carboximidamide (7-formamidino-7-deazaguanosine), which is a non-purine, non-pyrimidine ribonucleoside; its structure thus reflects extensive modification beyond the guanine-15 specified by corresponding gene sequences. The structure was established by mass spectrometry, and in particular from collision-induced dissociation mass spectra of derivatives formed by microscale permethylation, and is confirmed by chemical synthesis.