The formulation of the culture medium for a Chinese hamster ovary (CHO) cell line has been investigated in terms of the simultaneous replacement of glucose and glutamine, the most commonly employed carbon and nitrogen sources, pursuing the objective of achieving a more efficient use of these compounds, simultaneously avoiding the accumulation of lactate and ammonium in the medium. The key factor in this process is the selection of compounds that are slowly metabolized. Among the different compounds studied, galactose and glutamate provide the best results, allowing support of cell growth with an optimal balance between nutrient uptake and cell requirements and the generation of minimal quantities of lactate and ammonium. The attained results also highlight the capacity of the cells to redistribute their metabolism as a response to the changes in medium composition.