A detailed reaction network of mammalian cell metabolism contains hundreds of enzymatic reactions. By grouping serial reactions into single overall reactions and separating overlapped pathways into independent reactions, the total number of reactions of the network is significantly reduced. This strategy of manipulating the reaction network avoids the manipulations of a large number of reactions otherwise needed to determine the reaction extents. A stoichiometric material balance model is developed based on the stoichiometry of the simplified reaction network. Closures of material balances on glucose and each of the 20 amino acids are achieved using experimental data from three controlled fed-batch and one-batch hybridoma cultures. Results show that the critical role of essential amino acids, except glutamine, is to provide precursors for protein synthesis. The catabolism of some of the essential amino acids, particularly isoleucine and leucine, is observed when an excess amount of these amino acids is available in the culture medium. It was found that the reduction of glutamine utilization (for reducing ammonia production) is accompanied by an increase in the uptake of nonessential amino acids (NAAs) from the culture medium. This suggests that NAAs are necessary even though they are not essential for cell growth. A glutamine balance shows that less than 20% of the glutamine nitrogen is utilized for essential roles, such as protein and nucleotide syntheses. A relatively constant percentage (about 45%) of the glutamine nitrogen is utilized for NAA biosynthesis, despite the fact that the absolute amount varies among the four experiments. As to the carbon skeleton of glutamine, a significant portion enters the tricarboxylic acid (TCA) cycle. A material balance on glucose shows that most of the glucose (81%) is converted into lactate when glucose is in excess. On the other hand, when glucose is limited, lactate production is considerably reduced, while a major portion of glucose (48%) enters the TCA cycle. The fraction of glucose used for the synthesis of cellular components ranges from 9 to 28%.