In rat hepatocytes exposed to [2-13C]pyruvate, newly formed glucose was more efficiently labeled in the carbon C5 than C2, as well as in the carbon C6 than C1, suggesting enzyme-to-enzyme channeling of D-glyceraldehyde 3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase. Likewise the C1/C2 and C6/C5 ratios for 13C abundance in newly formed glucose, which largely exceeded the C3/C2 ratio of lactate or alanine and could reflect reversibility in the fumarase reaction, were compatible with the enzyme-to-enzyme tunneling of symmetrical Krebs cycle intermediates in the sequence of reactions catalyzed by succinyl-CoA synthetase, succinate dehydrogenase, and fumarase. This study further indicates that the major fraction of pyruvate is metabolized via pyruvate carboxylase rather than pyruvate dehydrogenase.