In mammalian cell cultures, ammonia that is released into the medium as a result of glutamine metabolism and lactate that is excreted due to incomplete glucose oxidation are both known to essentially inhibit the growth of cells. For some cell lines, for example, hybridoma cells, excreted ammonia also has an effect on product formation. Although glutamine has been generally considered as the major energy source for mammalian cells, it was recently found that various adherent cell lines (MDCK, CHO-K1, and BHK21) can grow as well in glutamine-free medium, provided glutamine is substituted with pyruvate. In such a medium the level of both ammonia and lactate released was significantly reduced. In this study, metabolic flux analysis (MFA) was applied to Madin Darby Canine Kidney (MDCK) cells cultivated in glutamine-containing and glutamine-free medium. The results of the MFA allowed further investigation of the influence of glutamine substitution with pyruvate on the metabolism of MDCK cells during different growth stages of adherent cells, e.g., early exponential and late contact-inhibited phase. Pyruvate seemed to directly enter the TCA cycle, whereas most of the glucose consumed was excreted as lactate. Although the exact mechanisms are not clear so far, this resulted in a reduction of the glucose uptake necessary for cellular metabolism in glutamine-free medium. Furthermore, consumption of ATP by futile cycles seemed to be significantly reduced when substituting glutamine with pyruvate. These findings imply that glutamine-free medium favors a more efficient use of nutrients by cells. However, a number of metabolic fluxes were similar in the two cultivations considered, e.g., most of the amino acid uptake and degradation rates or fluxes through the branch of the TCA cycle converting alpha-ketoglutarate to malate, which is responsible for the mitochondrial ATP synthesis. Besides, the specific rate of cell growth was approximately the same in both cultivations. Thus, the switch from glutamine-containing to glutamine-free medium with pyruvate provided a series of benefits without dramatic changes of cellular metabolism.