Although much information is available on in vitro role of ATP in regulation, the in vivo kinetics of reactions in which ATP plays a role are only partly known. In order to study such reactions, it is therefore necessary to study the role of ATP in vivo. This study presents an in vivo, targeted perturbation of the ATP flux in aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae, which was accomplished by transiently (20 min) changing the extracellular undissociated benzoic acid concentration via the pH of the culture. The performed pH shifts resulted in, within about 20 s, a 40% decrease (pH upshift) or a 23% increase (pH downshift) of the calculated ATP consumption rate while the specific glucose uptake rate did not change because of the glucose-limited condition. The pH upshift resulted in a strong decrease in the glycolytic and TCA cycle fluxes; carbon and energy balances indicated an increased flux toward storage carbohydrates. As expected, the pH downshift leads to the opposite effects. Overall, consistent responses were observed in the metabolic fluxes, the off gas concentrations of O-2 and CO2 and intracellular metabolite concentrations, except for the concentrations of adenosine nucleotides which unexpectedly only showed minor dynamics. This demonstrates that our knowledge of the regulation of the ATP level, the storage metabolism, and central carbon metabolism of yeast is still incomplete. The new dynamic metabolite datasets obtained in this study will prove of great value in developing kinetic models.