Queuosine (Q) [7-(((4,5-cis-dihydroxy-2-cyclopenten-1-yl)amino)methyl)-7-deaz agu anosine] usually occurs in the first position of the anticodon of tRNAs specifying the amino acids asparagine, aspartate, histidine, and tyrosine. The hypermodified nucleoside is found in eubacteria and eucaryotes. Q is synthesized de novo exclusively in eubacteria; for eucaryotes the compound is a nutrient factor. In Escherichia coli the Q precursor (oQ), carrying a 2,3-epoxy-4,5-dihydroxycyclopentane ring, is formed from tRNA precursors containing 7-(aminomethyl)-7-deazaguanine (preQ1) by the queA gene product. A genomic queA mutant accumulating preQ1 tRNA was constructed. The QueA enzyme was overexpressed as a fusion protein with the glutathione S-transferase from Schistosoma japonicum and purified to homogeneity by affinity and anion-exchange chromatography. The enzyme QueA synthesizes oQ from preQ1 in a single S-adenosylmethionine- (AdoMet-) requiring step, indicating that the ribosyl moiety of AdoMet is transferred and isomerized to the epoxycyclopentane residue of oQ. The identity of oQ was verified by HPLC and directly combined HPLC/mass spectrometry. The formation of oQ was reconstituted in vitro, applying a synthetic RNA. A 17-nucleotide microhelix (corresponding to the anticodon stem and loop of tRNA(Tyr) from E. coli) is sufficient to act as the RNA substrate for oQ synthesis. We propose that QueA is an S-adenosylmethionine:tRNA ribosyltransferase-isomerase.