The regulation of glycolytic genes in response to carbon source in the yeast Saccharomyces cerevisiae has been studied. When the relative levels of each glycolytic mRNA were compared during exponential growth on glucose or lactate, the various glycolytic mRNAs were found to be induced to differing extents by glucose. No significant differences in the stabilities of the PFK2, PGK1, PYK1, or PDC1 mRNAs during growth on glucose or lactate were observed. PYK::lacZ and PGK::lacZ fusions were integrated independently into the yeast genome at the ura3 locus. The manner in which these fusions were differentially regulated in response to carbon source was similar to that of their respective wild-type loci. Therefore, the regulation of glycolytic mRNA levels is mediated at the transcriptional level. When the mRNAs are ordered with respect to the glycolytic pathway, two peaks of maximal induction are observed at phosphofructokinase and pyruvate kinase. These enzymes (i) catalyze the two essentially irreversible steps on the pathway, (ii) are the two glycolytic enzymes that are circumvented during gluconeogenesis and hence are specific to glycolysis, and (iii) are encoded by mRNAs that we have shown previously to be coregulated at the translational level in S. cerevisiae (P. A. Moore, A. J. Bettany, and A. J. P. Brown, NATO ASI Ser. Ser. H Cell Biol. 49:421-432, 1990). This differential regulation of glycolytic mRNA levels might therefore have a significant influence upon glycolytic flux in S. cerevisiae.