The heat shock transcription factor (HSF) is a key regulator of the heat shock response. In Saccharomyces cerevisiae, the transcription activating ability of Hsf1 is repressed by its DNA-binding domain, but the detailed mechanism by which the inhibitory function is relieved in response to stress remains unknown. In this study, we isolated and characterized three hsf1 mutants with temperature-sensitive mutations in the DNA-binding domain. Two mutations inhibited DNA-binding activity, leading to decreased expression of target genes. The third mutation caused transcriptional defects without affecting DNA binding, and its suppressor mutation was located in a region important for sensing heat shock. These results indicate that the DNA-binding domain regulates both the DNA-binding and transcriptional activities of Hsf1, and suggest that these functions are located within discrete regions of the DNA-binding domain. (c) 2006 Elsevier Inc. All rights reserved.