Despite a wealth of information about the structure and composition of steroid receptors and their functional domains, little is known about the role of accessory proteins as mediators of receptor activity. To better define the role of such proteins in estrogen receptor (ER) function, we have used immunoaffinity, steroid affinity, and site-specific DNA-affinity chromatography to identify and characterize proteins that associate with human ER (hER) in extracts from MCF-7 cells and hER-expressing CHO (CHO-ER) cells. In addition to the expected 66-kDa hER, a 70-kDa protein was obtained and subsequently identified as a member of the heat shock protein family (hsp70). A 55-kDa protein, detected by all three approaches, was identified as a member of the protein disulfide isomerase family (PDI). Two proteins that were preferentially retained by an ER-specific DNA affinity column (p48 and p45) remain unidentified. Maximal interaction of purified hER with the vitellogenin A2 estrogen response element (ERE) occurred in the presence of all four associated proteins isolated by DNA-affinity chromatography. The increased stability of this complex was due primarily to an increase in the association rate of hER with ERE. Thus, accessory proteins may be required for optimal interaction of ER with EREs.