Molecular chaperones help nascent polypeptides fold correctly and multimeric protein complexes assemble productively, while minimizing the danger of aggregation in the protein-rich intracellular environment. Heat shock protein 90 (Hsp90) is an evolutionarily conserved molecular chaperone that participates in stabilizing and activating more than 200 proteins - referred to as Hsp90 ‘clients' - many of which are essential for constitutive cell signalling and adaptive responses to stress. To accomplish this task, Hsp90 and additional proteins termed co-chaperones form a dynamic protein complex known as the Hsp90 chaperone machine. The complex nature of Hsp90 regulation and the many ways in which it participates in cell physiology have been clarified in recent years, in part with the help of small molecule Hsp90 inhibitors. Considerable progress has been made in understanding the dynamic conformational flexibility of Hsp90 and in recognizing the importance of post-translational modifications in regulating Hsp90 function. Hsp90 function has been co-opted by cancer cells to preserve their survival in the face of numerous environmental insults, and growing clinical evidence suggests a benefit for targeting Hsp90, alone or in combination with other agents, in cancer.