The Q nucleosides isolated from rabbit liver tRNA are known to have sugars (mannose or galactose) linked to their cyclopentene diol moiety. A Q nucleoside containing mannose (manQ) was synthesized by a cell-free system from rat liver, using purified E. coli tRNAAsp as an acceptor and GDP-mannose as a donor molecule. The novel mannosyltransferase catalyzing this reaction was purified from a particulate-free soluble enzyme fraction and found to be strictly specific for tRNAAsp. These results, together with the anomeric configuration of mannose in Q nucleoside, indicate that no lipid intermediate is involved in the biosynthesis of Q nucleoside.