Considering the effects of pH on many aspects of cell metabolism, such as its role in signaling processes and enzyme kinetics, it is indispensable to include the measurement of the dynamics of the intracellular pH, when studying the fast dynamic response of cells to perturbations. It has been shown previously that the intracellular pH rapidly drops following an increase in external glucose concentration [Kresnowati, M.T.A.P., Suarez-Mendez, C., Groothuizen, M.K., Van Winden, W.A., Heijnen, J.J., 2007. Measurement of fast dynamic intracellular pH in Saccharomyces cerevisiae using benzoic acid pulse. Biotechnol. Bioeng. 97, 86-98; Ramos, S., Balbin, M., Raposo, M., Valle, E., Pardo, L.A., 1989. The mechanism of intracellular acidification induced by glucose in Saccharomyces cerevisiae. J. Gen. Microbiol. 135, 2413-2422; Van Urk, H., Schipper, D., Breedveld, G.J., Mak, P.R., Scheffers, W.A., Van Dijken, J.P., 1989. Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621. Biochim. Biophys. Acta 992(1), 78-86]. The mechanism for this fast intracellular acidification, however, has not been elucidated yet. This paper presents a metabolome-based analysis to reveal the physiological phenomena that cause the fast intracellular acidification following either a glucose pulse or an ethanol pulse to carbon-limited chemostat cultures of Saccharomyces cerevisiae. This quantitative study, which includes the determination of intracellular buffering capacity, the calculation of electric charge balance and the quantification of weak organic acid transport shows that none of the previously suggested mechanisms, i.e. increase in glucose phosphorylation and accumulation of CO(2), is sufficient to explain the measured decrease in intracellular pH following a glucose pulse.