In vivo kinetics of Saccharomyces cerevisiae are studied, in a time window of 150 s, by analyzing the response of O-2 and CO2 in the fermentor off-gas after perturbation of chemostat cultures by metabolite pulses. Here, a new mathematical method is presented for the estimation of the in vivo oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) directly from the off-gas data in such perturbation experiments. The mathematical construction allows effective elimination of delay and distortion in the off-gas measurement signal under highly dynamic conditions. A black box model for the fermentor off-gas system is first obtained by system identification, followed by the construction of an optimal linear filter, based on the identified off-gas model. The method is applied to glucose and ethanol pulses performed on chemostat cultures of S. cerevisiae. The estimated OUR is shown to be consistent with the independent dissolved oxygen measurement. The estimated in vivo OUR and CER provide valuable insights into the complex dynamic behavior of yeast and are essential for the establishment and validation of in vivo kinetic models of primary metabolism. (C) 2003 Wiley Periodicals, Inc.