The yeast heat shock transcription factor gene, HSF1, has been isolated from an S. cerevisiae genomic expression library (in lambda gt11). The sequenced gene encodes an 833 amino acid protein having a mass of 93,218 daltons. Expression of specific DNA-binding activity in E. coli and of transcriptional activity in yeast confirmed the identity of the cloned gene. Southern analysis and gene-disruption experiments indicate that the heat shock transcription factor is encoded by a single-copy, essential gene. The DNA-binding domain was localized to a 118 amino acid region in the amino-terminal third of the protein. Inspection of the DNA-binding domain reveals no resemblance to any currently known secondary structural motifs implicated in DNA recognition and binding.