First of all, the aim of this paper is to systematically analyze the metabolism of mammalian cell cultures and reduce the complicated metabolic networks to a manageable macroscopic reaction scheme. Herein, the central carbon metabolism (glycolysis, TCA cycle and glutaminolysis) of glucose and amino acids was investigated whereas the synthetic metabolism of cell and antibody was simply represented with macroscopically balanced reactions. Without losing generality, perfusion and fed-batch cultures of two typical cell lines (hybridoma and GS-CHO) were carried out in bioreactor. With macroscopic balance analysis and metabolic flux analysis (stationary and dynamical), the metabolic networks were rationally reduced thanks to the experimental facts that most of the amino acids (except glutamine, glutamate, and alanine) contributed negligibly to the central carbon metabolism, i.e., they were allocated mainly to the syntheses. Finally, with the aid of analysis of elementary flux mode analysis and extreme pathways, nine macro reactions were derived with physiological definitions. Furthermore, the results from above analyses (MBA, MFA, EFM and EPs) support following conclusions: the respiratory quotient value in mammalian cell culture generally should be near one; several potential metabolic bottlenecks (pyruvate to acetyl-CoA, cytosol NADH translocation into mitochondrion, glutamate dehydrogenation into TCA) may control and regulate cellular metabolism. These results will benefit the future study of dynamic modeling for process monitoring and control. (C) 2013 Elsevier Ltd. All rights reserved.