In response to hyperthermia, heat shock transcription factor (HSF) activates transcription of a set of genes encoding heat shock proteins (HSPs). The promoter regions of HSP genes contain the HSF binding sequence called the heat shock element (HSE), which consists of contiguous inverted repeats of the sequence 5'-nGAAn-3' (where n is any nucleotide). We have constructed an hsf1 mutant of Saccharomyces cerevisiae and analyzed genome-wide changes in heat shock response in the mutant cells. The results have revealed that Hsf1 is necessary for heat-induced transcription of not only HSP but also genes encoding proteins involved in diverse cellular processes such as protein degradation, detoxification, energy generation, carbohydrate metabolism, and maintenance of cell wall integrity. Approximately half of the Hsf1-regulated genes lacked the typical HSE in their promoter regions. Instead, several of these genes have a novel Hsf1 binding sequence that contains three direct repeats of nTTCn (or nGAAn) interrupted by 5 bp. The number and spacing of the repeating units are critical determinants for heat-induced transcription as well as for recognition by Hsf1. In the yeast genome, the presence of the sequence is enriched in Hsf1-regulated genes, suggesting that it is generally used as an HSE in the Hsf1 regulon.