In Saccharomyces cerevisiae the enzymes of glycolysis make up 30–60% of the soluble cellular protein. The first indication that glycolytic enzyme genes were coordinately regulated came with the isolation of gcr1 mutants, which have severely reduced levels of most glycolytic enzymes. GCR1 encodes a positive transcriptional activator, Gcr1p, that binds at the UAS elements of glycolytic enzyme genes with the assistance of Rap1p. An important question concerning Gcr1p is the extent of its action in the cell. Using high-density DNA microarrays, we defined a limited set of genes that are dependent on Gcr1p. For these studies we used two isogenic strains, wild type and a gcr1 mutant grown in the permissive medium of YP supplemented with glycerol and lactic acid. RNA was collected from cells during steady-state growth and used to prepare labelled cDNA. The labelled cDNA was hybridized to DNA microarrays. ORFs that displayed threefold or higher hybridization to cDNA from wild-type cells accounted for 30% of the hybridization of the total transcriptome, whereas these same ORFs accounted for only 4% of the transcriptome of the gcr1 mutant. We identified approximately 50 ORFs using the threefold cutoff. These ‘GCR1-dependent genes’ were placed in three classes: (i) glycolytic enzyme genes; (ii) ORFs encoded by Ty elements; and (iii) genes whose expression was not previously known to be dependent on Gcr1p. Of the eight glycolytic enzyme activities known to be severely affected by gcr1 mutations, ten ORFs encoding seven of the activities were identified. Only PGI1 was not identified using the threefold cutoff; it displayed a twofold difference. In total 32 of the GCR1-dependent genes identified by microarry analysis were previously known, or were expected, to be dependent on GCR1. The expression of the remaining ORFs identified was not previously known to be dependent on GCR1. Genes encoding ribosomal proteins were not identified as GCR1 dependent, suggesting that Rap1p interacts with activators other than Gcr1p at ribosomal protein gene UAS elements. In summary, a limited set of genes is dependent on GCR1 for expression, but together they compose 30% of the yeast transcriptome. The genomic expression studies described here further establish Gcr1p as one of the most potent transcriptional activators.