Expression of the GAL1 gene in Saccharomyces cerevisiae is strongly repressed by growth on glucose. We show that two sites within the GAL1 promoter mediate glucose repression. First, glucose inhibits transcription activation by GAL4 protein through UASG. Second, a promoter element, termed URSG, confers glucose repression independently of GAL4. We have localized the URSG sequences responsible for glucose repression to an 87-base-pair fragment located between UASG and the TATA box. Promoters deleted for small (20-base-pair) segments that span this sequence are still subject to glucose repression, suggesting that there are multiple sequences within this region that confer repression. Extended deletions across this region confirm that it contains at least two and possibly three URSG elements. To identify the gene products that confer repression upon UASG and URSG, we have analyzed glucose repression mutants and found that the GAL83, REG1, GRR1, and SSN6 genes are required for repression mediated by both UASG and URSG. In contrast, GAL82 and HXK2 are required only for UASG repression. A mutation designated urr1-1 (URSG repression resistant) was identified that specifically relieves URSG repression without affecting UASG repression. In addition, we observed that the SNF1-encoded protein kinase is essential for derepression of both UASG and URSG. We propose that repression of UASG and URSG is mediated by two independent pathways that respond to a common signal generated by growth on glucose.