In transformed cell lines, glucose and glutamine transport into cells as well as glycolysis and glutaminolysis pathways occur at high rates. As a consequence, cells in culture show a poor exploration of these resources and produce excessive amounts of lactic acid and ammonium. In this work, a murine hybridoma cell line (KB-26.5) was cultivated in batch cultures with different concentrations of oxamate, an specific inhibitor of lactate dehydrogenase. The results show that the conversion of pyruvate into lactate is absolutely necessary for cell survival as a way to reoxidize the large amounts of NADH produced in the rapid glycolysis; moreover, this metabolic analysis was completed with a continuous culture at changing inlet concentrations of glucose and glutamine. Steady-state viable cell, total cell, glucose, lactate, ammonium glutamine, and other amino acids were measured over a wide range of glucose and glutamine feed concentrations. The results show the metabolic deregulation existing in hybridoma cells and make it possible to calculate the minimal specific requirements for glucose (76.8 nmol 10(6) cells(-1) h(-1)) and glutamine (30.72 nmol 10(6) cells(-1) h(-1)) to maintain a cell population with minimal excretion of partly oxidized undesirable end products and a minimal increase in osmolarity. An explanation of these metabolic patterns is discussed in terms of NADH regeneration.