Abstract: Saccharomyces cerevisiae CYC1 gene expression has been studied in great detail with regard to the response to oxygen availability and carbon source. In the absence of oxygen and the presence of glucose, the CYC1 gene is completely repressed. Chromatin structure is thought to play an important role in CYC1 gene regulation, as nucleosome depletion results in 94-fold derepression. In addition, the CYC1 core promoter has been used extensively in hybrid constructs to study activation by heterologous transcription factors. Therefore, we set out to map the chromatin structure of the CYC1 promoter and determine its role in CYC1 gene regulation. We report here that the repressed CYC1 promoter contains no positioned nucleosomes over the core promoter. However, we did find TFIID and RNA polymerase II bound in a complex on the repressed promoter. These results indicate that recruitment of TFIID and RNA polymerase II are not rate-limiting steps in CYC1 activation.