In general, quantitative analysis of intracellular metabolic fluxes can be a useful tool for the investigation and understanding of mammalian cell physiology and its changes in response to environmental variations. To estimate these fluxes, a stoichiometric matrix taking into account the most relevant pathways involved in energy and mass transformations in a hybridoma cell line was constructed. This matrix, combined with the specific rates of consumption or production for the extracellular metabolites, was used to estimate the intracellular fluxes by means of a least squares procedure. To corroborate the estimated fluxes, experiments with C-13-labelled glucose were performed, and the C-12/C-13 ratio in the excreted lactate was determined experimentally by nuclear magnetic resonance (NMR). This ratio was compared to the predicted value using the calculated fluxes and showed good agreement.The interpretation of the results obtained allows two trends to be established in the metabolism of the cell line investigated. Firstly, the high rate of lactate lactate production in the hybridoma cell line studied may be due to the lack of activity of the malate-aspartate shuttle, meaning that the only way to oxidize quickly the NADH produced in the glycolysis was the synthesis of lactate. Secondly, the high consumption of glutamine unbalances the tricarboxylic acid cycle and the cell has to export some of the carbon excess in the TCA using the malate shunt.