The pentose phosphate pathway (PPP) was investigated in a rat model of hepatoma and results were compared to normal liver and other tissues. A novel and specific tracer of the PPP, [2,3-13C2]glucose, is introduced. The resulting isotopomers are informative because [1,2-13C2]lactate arises only from glycolysis and [2,3-13C2]lactate arises only from the PPP. The PPP was more active in the fed vs. fasted state in most tissues. These results correlated with mRNA expression of key enzymes in the PPP, and flux through both the PPP and glycolysis was substantially increased in hepatoma compared to healthy liver.