Unlike most chaperones, heat-shock protein 90 (Hsp90) interacts with a select group of ''client proteins'' that regulate essential biological processes. Little is known about how Hsp90 recognizes and binds these proteins. The glucocorticoid receptor (GR) is a well characterized Hsp90 client protein, whose hormone binding, nuclear-cytoplasmic trafficking, and transcriptional activity are regulated by Hsp90. Here, we provide evidence that unliganded and hormone-bound GR interact with two distinct, solventexposed hydrophobic sites in the Hsp90 C-terminal domain that contain the sequences ''MxxIM'' (HM10) and ''L/MxxIL'' (HM9). Our results indicate that binding of Hsp90 HM10 to unliganded GR stabilizes the unliganded ligand-binding pocket of GR indirectly by promoting an intramolecular interaction between the C-terminal ␣-helix (H12) and a solvent-exposed hydrophobic groove in the GR ligand binding domain. In the presence of hormone, Hsp90 appears to bind the hydrophobic groove of GR directly by mimicking the interactions of GR with transcriptional coactivators. The identified interactions provide insights into the mechanisms that enable Hsp90 to regulate the activity of both unliganded and hormonebound GR and to sharpen the cellular response to hormone.binding sites ͉ heat-shock protein 90 ͉ steroid hormone receptors