A transition from aerobic to anaerobic metabolism occurs as mitogen-activated thymocytes undergo proliferation. Glucose utilization and lactate formation increases 18- and 38-fold, respectively, during proliferation. The absolute amount of 14CO2 production by pyruvate dehydrogenase remains constant, while 14CO2 production by the tricarboxylic acid cycle is reduced during transition from a resting to a proliferating state. Addition of 2,4-dinitrophenol, an agent uncoupling oxidative phosphorylation, and phenacinemethosulfate, an electron acceptor, provide evidence that the reduction of glucose oxidation in proliferating thymocytes is caused neither by limitation of the tricarboxylic acid cycle itself nor by an insufficient supply of ADP. Our data suggest that enhanced cytosolic regeneration of NAD+ by induction of the glycolytic enzymes during proliferation effectively competes with NADH transport and its subsequent oxidation in the mitochondria. Mitogen-stimulated rat thymocytes cultured in a conventional medium containing glucose induce their glycolytic enzymes 8-10-fold in the S phase of the cell cycle and divide within a culture period of 72 h. Replacement of glucose by glutamine, glutamine and ribose, or glutamine and uridine prevents glycolytic enzyme induction and thymocyte proliferation. The effect of glucose on glycolytic enzyme induction cannot be mimicked by 3-O-methylglucose or 2-deoxyglucose. In conclusion, glucose is required for proliferation and the glycolytic enzyme induction that mediates the transition from oxidative to glycolytic energy production during the G1/S transition of rat thymocytes.