The heat shock response is a highly conserved mechanism that allows cells to withstand a variety of stress conditions. Activation of this response is characterized by increased synthesis of heat shock proteins (HSPs), which protect cellular proteins from stress-induced denaturation. Heat shock transcription factors (HSFs) are required for increased expression of HSPs during stress conditions and can be found in complexes containing components of the Hsp90 molecular chaperone machinery, raising the possibility that Hsp90 is involved in regulation of the heat shock response. To test this, we have assessed the effects of mutations that impair activity of the Hsp90 machinery on heat shock related events in Saccharomyces cerevisiae. Mutations that either reduce the level of Hsp90 protein or eliminate Cpr7, a CyP-40-type cyclophilin required for full Hsp90 function, resulted in increased HSF-dependent activities. Genetic tests also revealed that Hsp90 and Cpr7 function synergistically to repress gene expression from HSF-dependent promoters. Conditional loss of Hsp90 activity resulted in both increased HSF-dependent gene expression and acquisition of a thermotolerant phenotype. Our results reveal that Hsp90 and Cpr7 are required for negative regulation of the heat shock response under both stress and nonstress conditions and establish a specific endogenous role for the Hsp90 machinery in S. cerevisiae.