We analyzed the effects of site-directed mutations in the SUC2 promoter of Saccharomyces cerevisiae. Analyses were performed in wild-type as well as mig1 and tup1 mutant strains after the promoter mutants were reintroduced into the native SUC2 locus on the left arm of chromosome IX. Mutation of the two GC boxes revealed that these elements play two distinct roles: they are, as expected, required for Mig1-mediated repression but they are also necessary for activation of the SUC2 promoter in response to glucose limitation. The individual GC boxes are functionally redundant with regard to Mig1-mediated repression, however, only the upstream GC box is essential for high level expression of SUC2. Microccocal nuclease sensitivity of the SUC2 promoter in derepressed cells was reduced in the GC box mutant promoters, particularly in the vicinity of the TATA box. The difference in nuclease sensitivity between wild-type and GC box mutant promoters was not evident in tup1- cells. The formation of nuclease-resistant chromatin does not require the GC boxes, indicating that other cis-acting elements can serve to recruit the Ssn6-Tup1 co-repressor complex to the SUC2 promoter.