In order to directly determine the amount of label exchange that occurs in the tricarboxylic cycle from labeled alanine and lactate after the ingestion of a glucose load [1-13C]glucose was administered by continuous intraduodenal infusion to awake catheterized rats to achieve steady state jugular venous glycemia (160 mg/dl) for 180 min. Liver was freeze-clamped at 90 and 180 min, and perchloric acid extracts of the liver were subjected to 13C and 1H nuclear magnetic resonance analysis. Dilution in the oxaloacetate pool was determined by comparing the intrahepatic 13C enrichments of C2, C3 positions of glutamate with the C2, C3 positions of alanine and lactate. In addition steady state flux equations were derived for calculation of relative fluxes through pyruvate dehydrogenase/TCA cycle flux and pyruvate kinase flux/total pyruvate utilization. After glucose ingestion in a 24-h fasted rat direct conversion of glucose was responsible for 34% of glycogen. The intrahepatic dilution factor for labeled pyruvate in the oxaloacetate pool was 2.4. Using this factor, alanine and lactate contributed approximately 55% to glycogen formation. Pyruvate dehydrogenase flux ranged between 24 and 35% of total acetyl-coenzyme A (CoA) production and pyruvate kinase flux relative to total pyruvate utilization was approximately 40%.