Deuteration of 13C-enriched glucose has been used to prolong T1 of hyperpolarized 13C with the objective of monitoring glucose metabolism to lactate. However, the effect of deuteration on the flux in glycolysis in vivo has not been investigated extensively. Deuterated glucose was studied in perfused hearts and extracts were analyzed for a deuterium kinetic isotope effect using 1H, 13C and 2H NMR spectroscopy. Unexpectedly, there was extensive exchange of methyl deuterons in lactate with aqueous medium. Perdeuteration slows metabolism of glucose to lactate.