A network model for the determination of tumor metabolic fluxes from 13 C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mM [1,6-13 C 2 ]glucose under normoxic conditions at 37°C and monitored by 13 C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ϳ50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ϳ6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mM) and euglycemic (5 mM) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism.Cancer cells exhibit metabolic patterns that differ significantly from those of terminally differentiated adult tissues. They require rapid production of both energy and biosynthetic precursors to sustain a high rate of proliferation. The network of biochemical pathways underlying these processes is complex. Most of the individual enzymatic conversions involved in this network have probably already been identified, but a comprehensive and quantitative description of the fluxes involved is lacking. Methods to produce such descriptions will likely be very useful in the design of new therapeutics that specifically target the metabolic abnormalities of cancer as well as in clinical implementation of non-invasive methods for detection of cancer and its therapeutic response.The kinetics of 13 C isotope labeling as monitored by NMR spectroscopy has been used to measure flux through various metabolic pathways of perfused isolated cells and organs and in vivo (1-4). Kinetic data provide estimates of absolute flux, whereas steady-state data measure relative flux. Both types of analysis require validated mathematical models, which have been formulated for the heart (5-10), liver (11-13), brain (14 -16), a...