The pathways of glycerol-3-phosphate (G3P) generation for glyceride synthesis were examined in precision-cut liver slices of fasted and diabetic rats. The incorporation of 5mM [U¹⁴C]glucose into glyceride-glycerol, used to evaluate G3P generation via glycolysis, was reduced by ~26-36% in liver slices of fasted and diabetic rats. The glycolytic flux was reduced by ~60% in both conditions. The incorporation of 1 mM [2-¹⁴C]pyruvate into glyceride-glycerol (glyceroneogenesis) increased ~50% and ~36% in slices of fasted and diabetic rats, respectively, which also showed a two-fold increase in the activity phosphoenolpyruvate carboxykinase. The increased incorporation of 1.0 mM [2-¹⁴C]pyruvate] into glyceride-glycerol by slices of fasted rats was not affected by the addition of 5 mM glucose to the incubation medium The activity of glycerokinase and the incorporation of 1 mM [U-¹⁴C]glycerol into glyceride-glycerol, evaluators of G3P formation by direct glycerol phosphorylation, did not differ significantly from controls in slices of the two experimental groups. Rates of incorporation of 1 mM [2-¹⁴C]pyruvate and [U-¹⁴C]glycerol into medium glucose (gluconeogenesis) were ~140 and ~20% higher in fasted and diabetic slices than in controls. It could be estimated that glyceroneogenesis by liver slices of fasted rats contributed with ~20% of G3P generated for glyceride-glycerol synthesis, the glycolytic pathway with ~5% and direct phosphorylation of glycerol by glycerokinase with ~75%. Pyruvate contributed with 54% and glycerol with 46% of gluconeogenesis. The present data indicate that glyceroneogenesis has a significant participation in the generation of G3P needed for the increased glyceride-glycerol synthesis in liver during fasting and diabetes.