Primary metabolism of a murine hybridoma was probed with C-13 nuclear magnetic resonance (NMR) spectroscopy. Cells cultured in a hollow fiber bioreactor were serially infused with [1-C-13] glucose, [2-C-13] glucose, and [3-C-13] glutamine. In vivo spectroscopy of the culture was used in conjunction with off-line spectroscopy of the medium to determine the intracellular concentration of several metabolic intermediates and to determine fluxes for primary metabolic pathways. Intracellular concentrations of pyruvate and alanine were very high relative to levels observed in normal quiescent mammalian cells. Estimates made from labeling patterns in lactate indicate that 76% of pyruvate is derived directly from glycolysis; some is also derived from the malate shunt, the pyruvate/malate shuttle associated with lipid synthesis and the pentose phosphate pathway. The rate of formation of pyruvate from the pentose phosphate pathway was estimated to be 4% of that from glycolysis; this value is a lower limit and the actual value may be higher. Incorporation of pyruvate into the tricarboxylic acid (TCA) cycle appears to occur through only pyruvate dehydrogenase; no pyruvate carboxylase activity was detected. The malate shunt rate was approximately equal to the rate of glutamine uptake. The rate of incorporation of glucose-derived acetyl-CoA into lipids was 4% of the glucose uptake rate. The TCA cycle rate between isocitrate and alpha-ketoglutarate was 110% of the glutamine uptake rate.