1. The effect of incorporating D2O into the incubation medium on glycolysis and gluconeogenesis by hepatocytes from fasted rats was examined. 2. The substitution by heavy water, D2O, at concentrations from 10 to 40%, stimulated glucose uptake, lactate production and CO2 yields from glucose. At 10 mM glucose, 40% D2O doubled glucose uptake, increased CO2 production by 40%, and increased lactate production by 350%. 3. The stimulation of lactate production decreased at higher glucose concentrations, but was still substantial even at 80 mM glucose. 4. There was no effect on CO2 production above glucose concentrations of 30 mM. 5. Ten percent D2O showed little inhibition of lactate uptake, its oxidation and gluconeogenesis. At 40% D2O the inhibition ranged from 10 to 20%. 6. No effect of D2O on the rate of glucokinase or glucose-6-phosphatase was observed. 7. The concentration of fructose, 2,6-P was not affected by D2O.