Methods are presented for the preparation of a variety of -fructose phosphates, ¹³C-substituted at any single carbon site or at any two symmetrically disposed carbon sites, from either ¹³C-substituted pyruvate or -alanine. It is demonstrated that millimole quantities of product can be obtained in good yield following a “one-pot” incubation of ¹³C-substituted precursors with commercially available enzymes and cofactors of the glycolytic pathway. Since it has previously been shown that a wide variety of aldehydes serve as acceptable substrates for the final rabbit muscle aldolase-catalyzed condensation step, the method can potentially be applied to prepare a wide variety of ¹³C-substituted sugars and sugar phosphates.