A network model has been developed for analysis of tumor glucose metabolism from (13)C MRS isotope exchange kinetic data. Data were obtained from DB1 melanoma cells grown on polystyrene microcarrier beads contained in a 20-mm diameter perfusion chamber in a 9.4 T Varian NMR spectrometer; the cells were perfused with 26 mM [1,6-(13)C(2)]glucose under normoxic conditions and 37°C and monitored by (13)C NMR spectroscopy for 6 h. The model consists of ∼150 differential equations in the cumomer formalism describing glucose and lactate transport, glycolysis, TCA cycle, pyruvate cycling, the pentose shunt, lactate dehydrogenase, the malate-aspartate and glycerophosphate shuttles, and various anaplerotic pathways. The rate of oxygen consumption (CMRO(2)) was measured polarographically by monitoring differences in pO(2). The model was validated by excellent agreement between model predicted and experimentally measured values of CMRO(2) and glutamate pool size. Assuming a P/O ratio of 2.5 for NADH and 1.5 for FADH2, ATP production was estimated as 46% glycolytic and 54% mitochondrial based on average values of CMRO(2) and glycolytic flux (two experiments).