In this work, we used two approaches to localize the 90-kDa heat shock protein (hsp90)-binding site within the hormone-binding domain of the glucocorticoid receptor. In the first approach, derivatives of the glucocorticoid receptor deleted for increasing portions of the COOH terminus were translated in rabbit reticulocyte lysate, and the [35S]methionine-labeled translation products were immunoadsorbed with the 8D3 monoclonal antibody against hsp90. The data suggest that a segment from amino acids 604 to 659 (mouse) of the receptor is required for hsp90 binding. We have recently shown that the internal deletion mutant of the mouse receptor (delta 574-632) binds hsp90, although the complex is somewhat unstable (Housley, P. R., Sanchez, E. R., Danielsen, M., Ringold, G. M., and Pratt, W. B. (1990) J. Biol. Chem. 265, 12778-12781). The two observations indicate that amino acids 574-659 are involved in forming a stable receptor-hsp90 complex and that region 632-659 is especially important. To test this hypothesis directly, we synthesized three peptides corresponding to segments in region 624-665 and three peptides spanning the highly conserved sequence at amino acids 582-617, and we then tested the ability of the peptides to compete for the association of hsp90 with the L cell glucocorticoid receptor. In this assay, the immunopurified hsp90-free mouse receptor is incubated with rabbit reticulocyte lysate, which directs the association of rabbit hsp90 with the mouse receptor, simultaneously converting the receptor to the steroid binding state. All three peptides spanning region 624-665 and a peptide corresponding to segment 587-606 inhibited both hsp90 association with the receptor and reconstitution of steroid binding capacity. The data from all of the approaches support a two-site model for the hsp90-binding site in which the critical contact site occurs in region 632-659, which contains a short proline-containing hydrophobic segment and adjacent dipole-plus-cysteine motif that are conserved among all of the hsp90-binding receptors in the superfamily. A second hsp90 contact site is predicted in region 574-632, which contains the only highly conserved amino acid sequence in the receptor superfamily outside of the DNA-binding domain.