Lack of sufficient quantities of isotopically labeled materials has precluded the use of heavy atom isotope effects to investigate mechanisms of nucleotidyl transfer reactions in nucleic acids. Here we achieve regioselective opening of 2,2'-cyclouridine with [(18)O2]benzoic acid/potassium hydride, allowing an efficient "one-pot" synthesis of [2'-18O]uridine in 88% yield. Conversion to the corresponding phosphoramidite enables solid-phase synthesis of [2'-(18)O] RNA substrates for isotope effect studies with nucleotidyl transferases and hydrolases.