A global kinetic study of the central metabolism of Vero cells cultivated in a serum-free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum-free medium which is a valuable help for the implementation of this cell line in serum-free industrial production processes. Biotechnol. Bioeng. 2010; 107: 143-153. (C) 2010 Wiley Periodicals, Inc.