tRNA–guanine transglycosylase (TGT) is a key enzyme involved in the posttranscriptional modification of tRNA across the three kingdoms of life. In eukaryotes and eubacteria, TGT is involved in the introduction of queuine into the anticodon of the cognate tRNAs. In archaebacteria, TGT is responsible for the introduction of archaeosine into the D-loop of the appropriate tRNAs. The tRNA recognition patterns for the eubacterial (Escherichia coli) TGT have been studied. These studies are all consistent with a restricted recognition motif involving a U-G-U sequence in a seven-base loop at the end of a helix. While attempting to investigate the potential of negative recognition elements in noncognate tRNAs via the use of chimeric tRNAs, we have discovered a second recognition site for the E. coli TGT in the TΨC arm of in vitro-transcribed yeast tRNAPhe. Kinetic analyses of synthetic mutant oligoribonucleotides corresponding to the TΨC arm of the yeast tRNAPhe indicate that the specific site of TGT action is G53 (within a U-G-U sequence at the transition of the TΨC stem into the loop). Posttranscriptional base modifications in tRNAPhe block recognition by TGT, most likely due to a stabilization of the tRNA structure such that G53 is inaccessible to TGT. These results demonstrate that TGT can recognize the U-G-U sequence within a structural context that is different than the canonical U-G-U in the anticodon loop of tRNAAsp. Although it is unclear if this second recognition site is physiologically relevant, this does suggest that other RNA species could serve as substrates for TGT in vivo.