Commonly steady state analysis of microbial metabolism is performed under well defined physiological conditions in continuous cultures with fixed external rates. However, most industrial bioprocesses are operated it? fed-batch mode under non-stationary conditions, which cannot be realized in chemostat cultures. A novel experimental setup-rabid media transition-enables steady state perturbation of metabolism on a time scale of several minutes it? parallel to operating bioprocesses. For this purpose, cells are separated from the production process and transferred into a lab-scale stirred-tank reactor with modified environmental conditions. This new approach was evaluated experimentally in four rapid media transition experiments with Escherichia coli from a fed-batch process. We tested the reaction to different carbon sources entering at various points of central metabolism. In all cases, the applied substrates (glucose, succinate, acetate, and pyruvate) were immediately utilized by the cells. Extracellular rates and metabolome data indicate a metabolic steady state during the,short-term cultivation. Stoichiometric analysis revealed distribution of intracellular fluxes, which differs drastically subject to the applied carbon source. For some reactions, the variation of flux could be correlated to changes of metabolite concentrations. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1-10, 2010