The upstream activating sequence (UASG) of the adjacent and divergently transcribed GAL1 and GAL10 promoters of Saccharomyces cerevisiae regulates the induction of the corresponding genes in response to the presence of galactose. We constructed chimeric yeast promoters in which a different UAS, UASC from the iso-1-cytochrome c (CYC1) gene of S. cerevisiae, was fused at different locations upstream of GAL1 (UASC-GAL1 promoters) or GAL10 (UASC-GAL10 promoters) and used to monitor the activity of UASG in cells grown in the presence or absence of galactose. Though the CYC1 promoter is fully induced in yeast grown in glycerol medium, UASC-GAL chimeric promoters containing UASG were repressed as much as 400-fold (UASC-GAL1) or 1350-fold (UASC-GAL10) in this growth medium. Several distinct portions of the GAL1-GAL10 divergent promoter region blocked the UASC-induced expression of the GAL1 and GAL10 promoters, whereas others did not, suggesting that several distinct negative control elements are present that may repress transcription of GAL1 and GAL10 in the absence of galactose. The approximate locations of these negative control elements were delimited to sites adjacent to or possibly overlapping the sites at which the positive control protein GAL4 binds in UASG. Deletion derivatives of GAL4 that fail to induce transcription from the wild-type GAL promoters but retain the DNA binding domain significantly derepressed the expression of the UASC-GAL chimeric promoters. These results, combined with those of earlier studies, suggest the possibility that GAL4 normally induces transcription of GAL1 and GAL10 by blocking the activity of these negative control elements, in addition to stimulating transcription by a mechanism of positive control.