To better define the role of accessory protein as mediators of estrogen receptor (ER) function, we have used immuno-, steroid-, and site-specific DNA-affinity chromatography to identify and characterize proteins that associate with ER in extracts from ER-expressing Chinese hamster ovary (CHO-ER) cells. Two associated proteins [70 and 55 kilodaltons (kDa)] were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis silver stain analysis of all three column eluates. Two additional proteins (45 and 48 kDa) were preferentially retained by the ER-specific DNA affinity column. The 70-kDa protein was subsequently identified by Western blot analysis as a heat shock protein (hsp70). The 55-kDa protein was identified by N-terminal microsequencing as a member of the protein disulfide isomerase family. The 48- and 45-kDa proteins remain unidentified. To determine the possible differential effects of estrogen agonists and antagonists on human (h) ER interaction with these proteins, CHO-ER cells were labeled with [35S]methionine in the presence of estradiol, 4-hydroxytamoxifen (OH-Tam; partial agonist/antagonist), or ICI 182,780 (complete antagonist). None of these ligands altered the pattern of associated proteins when hER complexes were isolated by adsorption to the vitellogenin A2 estrogen response element (ERE). However, when hER was isolated by immunoadsorption, a reduction in the level of associated hsp70 was observed following treatment with estradiol or OH-Tam, compared to no treatment or treatment with ICI 182,780. By gel retardation analysis, maximal interaction of affinity-purified hER with ERE occurred in the presence of all four associated proteins. Removal of the 48- and 45-kDa proteins and/or hsp70 resulted in a decrease in hER/ERE association, which could be restored by the addition of purified hsp70 and/or a mixture of the 48- and 45-kDa proteins. The increased stability of the restored complex was due primarily to an increase in the association rate of hER with ERE. These results suggest that accessory proteins may be required for maximal ER interaction with EREs and that estrogens and estrogen antagonists may promote differential retention of hsp70 in the presence or absence of a specific ERE.