AbstractDifferent methods for oxygen uptake rate (OUR) determinations in animal cell cultivation were investigated using a high quality mass spectrometer.Dynamic measurementshave considerable disadvantages because of disturbances of the growing cells by the necessary variations of dissolved oxygen concentration. Only infrequent discrete measurements are possible using this method.Stationary liquid phase balanceyielded better results with much higher frequency. Gas phase balancing has the advantage of not requiring dissolved oxygen measurement and knowledge ofKLa, both of them are easily biased. It was found that simple gas phase balancing is either very inaccurate (error larger than expected signal) or very slow, with gas phase residence times of several hours. Therefore, anew method of aerationwas designed. Oxygen and CO2transfer are mainly achieved via sparging. The gas released to the headspace is diluted with a roughly 100‐fold stream of an inert gas (helium). Through this dilution, gas ratios are not changed for O2, CO2, Ar, and N2. The measurement of lower concentrations (parts per million and below) is easy using mass spectrometry with a secondary electron multiplier. With this new method an excellent accuracy and sufficient speed of analysis were obtained. All these on‐line methods for OUR measurement were tested during the cultivation of animal cells. The new method allowed better study of the kinetics of animal cell cultures as was shown with a hybridoma cell line (HFN 7.1, ATCC CRL 1606) producing monoclonal antibodies against human fibronectin. With the aid of these methods it was possible to find a correlation between a rapid decrease in oxygen uptake rate (OUR) and glutamine concentration. The sudden decrease in OUR can be attributed to glutamine depletion. This provided a basis for the controlled addition of glutamine to reduce the formation of ammonia produced by hydrolysis. This control method based on OUR measurement resulted in increased cell concentration and threefold higher product concentration. © 1995 John Wiley&Sons,