Hybridoma cells were,cultivated in a chemically defined medium in continuous cultures. These cultures reached different steady states marked by distinctive cell metabolism depending on the culture conditions leading to the steady state. Those steady states with different metabolism are characterized by different stoichiometric ratios of lactate production to glucose consumption (DeltaL/DeltaG). The specific consumption rates of glucose, glutamine and other amino acids are reduced when DeltaL/DeltaG reduces. Those steady states do not have a few discrete values of DeltaL/DeltaGs, rather they span from a high DeltaL/DeltaG state (> 1.0) to an intermediate state (0.1 less than or equal to DeltaL/DeltaG less than or equal to 1.0), and reduces even further at a low DeltaL/DeltaG state (< 0.1). Metabolic flux analysis was performed to compare energy metabolism of cells in cultures representing these three distinct metabolic states. The material balance on carbon and nitrogen was facilitated by the use of chemically defined medium. The formation of biomass was systematically estimated. It was revealed that all glycolysis and TCA cycle fluxes are reduced as DeltaL/DeltaG decreases. At the low DeltaL/DeltaG state, a reduction in amino acid specific consumption rate is accompanied by a reduction in all the fluxes around pyruvate. The analysis also shows that the outflux from the TCA cycle to form pyruvate, which contributes to lactate formation, is possibly linked to the higher consumption rate of amino acids at the high DeltaL/DeltaG state. Taken together the results suggest the amino acid metabolism plays an important role in reducing lactate production in mammalian cell culture.