Anaerobic and aerobic chemostat cultures of Saccharomyces cerevisiae were performed at a constant dilution rate of 0.10 h(-1). The glucose concentration was kept constant, whereas the nitrogen concentration was gradually decreasing; i.e., the conditions were changed from glucose and energy limitation to nitrogen limitation and energy excess. This experimental setup enabled the glycolytic rate to be separated from the growth rate. There was an extensive uncoupling between anabolic energy requirements and catabolic energy production when the energy source was present in excess both aerobically and anaerobically. To increase the catabolic activity even further, experiments were carried out in the presence of 5 mM acetic acid or benzoic acid. However, there was almost no effect with acetate addition, whereas both respiratory (aerobically) and fermentative activities were elevated in the presence of benzoic acid. There was a strong negative correlation between glycolytic flux and intracellular ATP content; i.e., the higher the ATP content, the lower the rate of glycolysis. No correlation could be found with the other nucleotides tested (ADP, GTP, and UTP) or with the ATP/ADP ratio. Furthermore, a higher rate of glycolysis was not accompanied by an increasing level of glycolytic enzymes. On the contrary, the glycolytic enzymes decreased with increasing flux. The most pronounced reduction was obtained for HXK2 and ENO1. There was also a correlation between the extent of carbohydrate accumulation and glycolytic flux. A high accumulation was obtained at low glycolytic rates under glucose limitation, whereas nitrogen limitation during conditions of excess carbon and energy resulted in more or less complete depletion of intracellular storage carbohydrates irrespective of anaerobic or aerobic conditions. However, there was one difference in that glycogen dominated anaerobically whereas under aerobic conditions, trehalose was the major carbohydrate accumulated. Possible mechanisms which may explain the strong correlation between glycolytic flux, storage carbohydrate accumulation, and ATP concentrations are discussed.