Understanding how a regulatory protein occupies its sites in vivo is central to understanding gene regulation. Using the yeast Gal4 protein as a model for such studies, we have found 239 potential Gal4 binding sites in the yeast genome, 186 of which are in open reading frames (ORFs). This raises the questions of whether these sites are occupied by Gal4 and, if so, to what effect. We have analyzed the Saccharomyces cerevisiae ACC1 gene (encoding acetyl-coenzyme A carboxylase), which has three Gal4 binding sites in its ORF. The plasmid titration assay has demonstrated that Gal4 occupies these sites in the context of an active ACC1 gene. We also find that the expression of the ACC1 is reduced fourfold in galactose medium and that this reduction is dependent on the Gal4 binding sites, suggesting that Gal4 bound to the ORF sites affects transcription of ACC1. Interestingly, removal of the Gal4 binding sites has no obvious effect on the growth in galactose under laboratory conditions. In addition, though the sequence of the ACC1 gene is highly conserved among yeast species, these Gal4 binding sites are not present in the Kluyveromyces lactis ACC1 gene. We suggest that the occurrence of these sites may not be related to galactose regulation and a manifestation of the "noise" in the occurrence of Gal4 binding sites.