The upstream activation site (UAS) of the yeast CYC1 gene is shown to contain two homologous subsites, UAS1 and UAS2. Each site, when placed upstream of the transcriptional initiation region of the yeast LEU2 gene, activates LEU2 transcription which is regulated by catabolite repression. UAS1 is responsible for most of the transcription under glucose repressed conditions, while UAS1 and UAS2 contribute equally to lactate derepressed transcription. A single point mutation in UAS2 increases its activity in glucose 10- to 20-fold. Several experiments indicate that UAS1 and UAS2 are regulated distinctly at the molecular level. First, UAS1 but not UAS2 is fully depressed in glucose by increasing the levels of intracellular heme. Second, trans-acting regulatory mutations, hap1-1 and hap2-1, selectively abolish the activity of UAS1 or UAS2. HAP1 appears to encode a protein that mediates catabolite repression of UAS1 by responding to intracellular heme levels.