Transcription of SUC2, the gene that encodes the cytoplasmic and secreted forms of the enzyme invertase, is controlled by glucose repression and derepression mechanisms in Saccharomyces cerevisiae. Several regulatory factors such as the Mig1p-Tup1p-Ssn6p repressor complex and the Snf1p kinase complex have been identified previously as regulators of SUC2 expression. We show that, in addition to these factors, expression of SUC2 is affected by mutations in the gene GCR1 that encodes the glycolysis regulatory protein Gcr1p. Expression of Suc2-LacZ was not repressed by glucose in gcr1 mutant yeast cells exposed to glucose. Furthermore, secreted invertase activity was constitutively expressed under glucose-repressed and derepressed conditions in gcr1 mutants. DNA gel mobility shift assays and in-vitro DNase I protection experiments mapped a DNA binding site for Gcr1p in the transcriptional control region of the SUC2 gene, next to a previously mapped Mig1p binding site. However, the mechanism by which gcr1 mutations relieve glucose repression remains obscure.