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Saccharomyces cerevisiae ATCC 24860 was cultivated in chemostat culture under anoxic conditions with 111.1 mmol of glucose liter ؊1 alone or with a mixture of 66.7 mmol of xylulose liter ؊1 and 111.1 mmol of glucose liter ؊1. The substrate consumption rate was 5.4 mmol g of cells ؊1 h ؊1 for glucose, whereas for xylulose it was 1.0 mmol g of cells ؊1 h ؊1. The ethanol yield decreased from 0.52 carbon mole of ethanol produced per carbon mole of sugar consumed during the utilization of glucose alone to 0.49 carbon mole produced per carbon mole consumed during the simultaneous utilization of xylulose and glucose, while cell biomass was maintained at 2.04 to 2.10 g liter ؊1. Xylulose coutilization was accompanied by a shift in product formation from ethanol to acetate and arabinitol. Xylulokinase activity was absent during glucose metabolism but detectable during simultaneous utilization of xylulose and glucose. Xylulose cometabolism resulted in increased in vitro activity of pyruvate decarboxylase and an increased concentration of the intracellular metabolite fructose 1,6-diphosphate without significant changes in the concentrations of 6-phosphogluconate and pyruvate. The results are discussed in relation to (i) altered enzyme activities and (ii) the redox flux of the cell.