Abstract5′-18O labeled RNA oligos are important probes to investigate the mechanism of 2′-O-transphosphorylation reactions. Here we describe a general and efficient synthetic approach to the phosphoramidite derivatives of 5′-18O labeled nucleosides starting from the corresponding commercially available 5′-O-DMT protected nucleosides. Using this method, we prepared 5′-18O-guanosine phosphoramidite in 8 steps (13.2% overall yield), 5′-18O-adenosine phosphoramidite in 9 steps (10.1% overall yield) and 5′-18O-2′-deoxyguanosine phosphoramidite in 6 steps (12.8% overall yield). These 5′-18O labeled phosphoramidites can be incorporated into RNA oligos by solid phase synthesis for determination of heavy atom isotope effects in RNA 2′-O-transphosphorylation reactions.