The first small molecule inhibitor of the molecular chaperone Heat Shock Protein 90 (HSP90) was identified more than 20 years ago. Upon determination of the drug binding site and clarification of its mechanism of action, HSP90 was validated as an anti-cancer molecular target, robust preclinical activity was demonstrated, and the first clinical trials of an HSP90 inhibitor in cancer patients were initiated several years later. Since then, additional natural product and synthetic HSP90 inhibitors have been identified and many have been clinically evaluated. As a class, these inhibitors are ATP-competitive and inhibit the ATPase driven HSP90 chaperone cycle, causing degradation of HSP90-dependent proteins (termed clients), including many oncogenes, via the proteasome. While numerous clinical trials have been performed with HSP90 inhibitors and encouraging data have been obtained, no HSP90 inhibitor has yet received FDA approval. One consequence of HSP90 inhibition is upregulation of the cytoprotective heat shock response, a broad transcriptional response to proteotoxic stress that is regulated by the transcription factor Heat Shock Factor 1 (HSF1). HSF1 also mediates a cancer-specific transcriptional program. HSP90 is proposed to sequester HSF1 in unstressed cells, but there is little evidence to support this model. A better mechanistic understanding of the role of HSP90 in modulating HSF1 may facilitate improved clinical activity of HSP90 inhibitors. We recently showed that HSP90 binding to HSF1 depends on HSP90 conformation and is only readily visualized for the ATP-dependent, N-domain dimerized chaperone, a conformation only rarely sampled by mammalian HSP90. Further, we show that ATP-competitive, N-domain targeted HSP90 inhibitors disrupt this interaction, resulting in the increased duration of HSF1 occupancy of the hsp70 promoter and significant prolongation of both constitutive and heat-induced HSF1 transcriptional activity. Not only do these suggest a role for HSP90 in feedback regulation of the proteotoxic stress response, but this characteristic of current HSP90 inhibitors likely contributes to lack of robust clinical response and we are currently evaluating several strategies that target HSP90 inhibitor effects on HSF1 without impacting HSP90 inhibitor-mediated degradation of oncogenic clients. Finally, a consistent property of HSP90 inhibitors to concentrate in tumors and to remain for an extended time period compared to normal tissues has prompted the use of HSP90 inhibitors as intracellular delivery vehicles to direct and concentrate conjugated cytotoxic agents in tumor while sparing normal tissues. This strategy extends the potential clinical utility of HSP90 inhibitors in cancer and a Phase 1 trial of such an HSP90-drug conjugate is underway.