Gluconeogenesis was measured quantitatively by mass isotopomer distribution analysis in rats in vivo through determination of the isotopic enrichment of the pool of hepatic triose phosphates, the immediate precursors of glucose, using primarily [2-13C]glycerol infusion after fasting, and following fructose and glucose infusion. The results showed that the liver controls gluconeogenesis, the metabolic sources and disposal of triose phosphates, and the contribution of glucose from glycogen in response to alterations in substrate availability.