A well-defined set of isogenic yeast strains has been constructed whereby each strain contains a different HXK2::lacZ gene fusion integrated at the URA3 locus. These HXK2::lacZ fusions differ in the amount of the HXK2 gene (encoding hexokinase 2 isoenzyme) that is fused to the lacZ reporter gene. Comparison of the beta-galactosidase activities of each strain during growth on glucose or ethanol revealed that some part of the coding region between +39 and +404 bp is involved in repressing gene expression in a carbon source dependent manner. A series of deletions of this HXK2 coding region were constructed and fused upstream of a minimal CYC1::lacZ promoter. beta-Galactosidase activities on glucose or ethanol growth yeast calls revealed that two different regulatory elements are present in this DNA region. Gel mobility shift analysis and in vitro DNase I footprinting have shown that proteins bind specifically to two downstream repressor sequences (DRS1 located from +140 to +163 and DRS2 located between +231 and +251) that influence the rate of HXK2 transcription when ethanol is used as carbon source by Saccharomyces cerevisiae. We identified and partially purified a 18 kDa protein that binds specifically to synthetic double-stranded oligonucleotides containing the (A/C)(A/G)GAAAT box sequence. Our data suggest that p18 synthesis is under the control of genes involved in glucose repression (MIG1 = CAT4) and glucose derepression (SNF1 = CAT1).