Several mutants of the human estrogen receptor (ER) were transiently expressed in Cos 7 cells in order to determine the regions involved in the formation of complexes with the heat shock protein Mr approximately 90,000 (hsp 90). The formation of the cytosol non-DNA binding 8-9 S complexes (8-9 S ER) was monitored by glycerol gradient ultracentrifugation. It was established that the N-terminal region of the receptor, including the two zinc fingers of the DNA binding domain (DBD), is not required for the formation of the 8-9 S ER complexes. Conversely, deletion of the entire ligand binding domain (LBD) produced truncated receptor mutants that are constitutive transcriptional activators and did not form 8-9 S ER complexes, confirming results obtained previously with the glucocorticosteroid receptor (Pratt, W.B., Jolly, D.J., Pratt, D.V., Hollenberg, S.M., Giguerre, V., Cadepond, F., Schweizer-Groyer, G., Catelli, M.G., Evans, R.M., and Baulieu, E.E. (1988) J. Biol. Chem. 263, 267-273). However, no limited subregion of the LBD was found to be uniquely involved in hsp 90 binding. A highly positively charged region situated at the C-terminal extremity of the DBD (between amino acids 251 and 271) also appeared to be implicated. Although not sufficient, this sequence is necessary for the formation of the 8-9 S ER; it also corresponds to the NL1 nuclear localization domain of steroid receptors. Taken together, these results suggest that the formation of complexes with hsp 90 involves several receptor regions, and they are consistent with the proposal that hsp 90 inhibits receptor function and can be released by hormone binding to the LBD.