BACKGROUND: Improvement of the mammalian cell culture process is based on the implementation of culture strategies with higher productivities, such as fed-batch or perfusion processes. The efficient development of these culture strategies depends strongly on the availability of suitable monitoring tools to determine the activity of cell culture and adjust nutrient feeding correspondingly. RESULTS: In this study oxygen uptake rate (OUR), cell density and glucose concentration on-line monitoring systems are applied to control fed-batch processes. Different combinations of these monitoring systems were used to determine cell growth and metabolic activity of cell culture in order to adjust substrate feeding in a balanced manner. Application of these monitoring tools in fed-batch processes allowed extension of the exponential growth of hybridoma cells up to 2-3-fold and increased antibody productivity from 84 to 168% with respect to conventional batch culture. CONCLUSIONS: The three control strategies tested have been successful in providing improvement of the fed-batch culture of the hybridoma cell line used. Among the different alternatives studied, the use of OUR on-line monitoring to simultaneously determine cell growth and metabolic activity appears to be the best method to adjust substrate feeding owing to its simplicity and robustness.