Growth temperature has a profound impact on the kinetic properties of enzymes in microbial metabolic networks. Activities of glycolytic enzymes in Saccharomyces cerevisiae were up to 7.5-fold lower when assayed at 12 degrees C than at 30 degrees C. Nevertheless, the in vivo glycolytic flux in chemostat cultures (dilution rate: 0.03 h(-1)) grown at these two temperatures was essentially the same. To investigate how yeast maintained a constant glycolytic flux despite the kinetic challenge imposed by a lower growth temperature, a systems approach was applied that involved metabolic flux analysis, transcript analysis, enzyme activity assays, and metabolite analysis. Expression of hexose-transporter genes was affected by the growth temperature, as indicated by differential transcription of five HXT genes and changed zero trans-influx kinetics of [(14)C]glucose transport. No such significant changes in gene expression were observed for any of the glycolytic enzymes. Fermentative capacity (assayed off-line at 30 degrees C), which was 2-fold higher in cells grown at 12 degrees C, was therefore probably controlled predominantly by glucose transport. Massive differences in the intracellular concentrations of nucleotides (resulting in an increased adenylate energy charge at low temperature) and glycolytic intermediates indicated a dominant role of metabolic control as opposed to gene expression in the adaptation of glycolytic enzyme activity to different temperatures. In evolutionary terms, this predominant reliance on metabolic control of a central pathway, which represents a significant fraction of the cellular protein of the organism, may be advantageous to limit the need for protein synthesis and degradation during adaptation to diurnal temperature cycles.