The yeast HIS3 promoter region contains two functionally distinct TATA elements, TC and TR, that are responsible respectively for initiation from the +1 and +13 sites. Both TC and TR support basal HIS3 transcription and require the TATA binding protein TFIID, but only TR responds to transcriptional activation by GCN4 and GAL4. By selecting for yeast strains that increase transcription by a GCN4 derivative with a defective activation domain, we have isolated a temperature-sensitive mutation in CDC39, a previously defined gene implicated in cell-cycle control and the pheromone response. This cdc39-2 mutation causes increased basal transcription of many, but not all genes, as well as increased transcriptional activation by GCN4 and GAL4. Surprisingly, basal HIS3 transcription from the +1 initiation site is strongly increased, while initiation from the +13 site is barely affected. Thus, unlike acidic activator proteins that function through TR, CDC39 preferentially affects transcription mediated by TC. CDC39 is an essential gene that encodes a very large nuclear protein (2108 amino acids) containing two glutamine-rich regions. These observations suggest that CDC39 negatively regulates transcription either by affecting the general RNA polymerase II machinery or by altering chromatin structure.