Abstract: The capability of bakers' yeast (Saccharomyces cerevisiae) to rapidly increase its glycolytic flux upon a switch from respiratory to fermentative sugar metabolism is an important characteristic for many of its multiple industrial applications. An increased glycolytic flux can be achieved by an increase of the glycolytic enzyme capacities (Vmax) and/or by changes of the concentrations of low-molecular weight substrates, products and effectors. The goal of the present study is to understand the time-dependent, multi-level regulation of glycolytic enzymes during a switch from fully respiratory to fully fermentative conditions. The switch from glucose-limited aerobic chemostat growth to full anaerobiosis and glucose excess caused a fast acceleration of fermentative metabolism. Although the capacities (Vmax) of the glycolytic enzymes did not change until 45 min after the switch, the intracellular levels of several substrates, products and effectors involved in the regulation of glycolysis did strongly change during the initial 45 min (e.g. build-up of phosphofructokinase activator fructose-2,6-bP). This study reveals two distinct phases in the upregulation of glycolysis upon a switch to fermentative conditions: i) an initial phase where regulation occurs completely through changes in metabolite levels and ii) a second phase where regulation is achieved through a combination of changes in Vmax and metabolite concentrations. This multi-level regulation study qualitatively explains the increase in flux through the glycolytic enzymes upon a switch of S. cerevisiae to fermentative conditions and provides a better understanding of the role of different regulatory mechanisms that influence the dynamics of yeast glycolysis.