Transcriptional activation of heat shock genes is a reversible and multistep process involving conversion of inactive heat shock factor 1 (HSF1) monomers into heat shock element (HSE)-binding homotrimers, hyperphosphorylation, and further modifications that induce full transcriptional competence. HSF1 is controlled by multiple regulatory mechanisms, including suppression by additional cellular factors, physical interactions with HSP70, and integration into different cellular signaling cascades. However, the signaling mechanisms by which cells respond to stress and control the HSF1 activation-deactivation pathway are not known. Here we demonstrate that HSP90, a cellular chaperone known to regulate several signal transduction molecules and transcription factors, functions in the regulation of HSF1. The existence of HSF1-HSP90 heterocomplexes was shown by coimmunoprecipitation of HSP90 with HSF1 from unshocked and heat-shocked nuclear extracts, recognition of HSF1-HSE complexes in vitro by using HSP90 antibodies (Abs), and recognition of HSF1 in vivo by HSP90 Abs microinjected directly into oocyte nuclei. The functional impact of HSP90-HSF1 interactions was analyzed by using two strategies: direct nuclear injection of HSP90 Abs and treatment of cells with geldanamycin (GA), an agent that specifically blocks the chaperoning activity of HSP90. Both HSP90 Abs and GA delayed the disassembly of HSF1 trimers during recovery from heat shock and specifically inhibited heat-induced transcription from a chloramphenicol acetyltransferase reporter construct under control of the hsp70 promoter. HSP90 Abs activated HSE binding in the absence of heat shock, an effect that could be reversed by subsequent injection of purified HSP90. GA did not activate HSE binding under nonshock conditions but increased the quantity of HSE binding induced by heat shock. On the basis of these findings and the known properties of HSP90, we propose a new regulatory model in which HSP90 participates in modulating HSF1 at different points along the activation-deactivation pathway, influencing the interconversion between monomeric and trimeric conformations as well as transcriptional activation. We also put forth the hypothesis that HSP90 links HSF1 to cellular signaling molecules coordinating the stress response.