Chromosomal proteins that impart high affinity and specificity to the binding of the estrogen receptor (ER) to DNA are termed estrogen receptor binding factors (ERBFs). Certain partially purified chromosomal protein fractions obtained from rabbit uterine chromatin by extraction with various molarities of GdnHCl when reconstituted to double-stranded DNA demonstrated high affinity binding for the ER. We report the purification and characterization of ERBFs in the chromosomal protein fraction extracted with 4 M GdnHCl (CP4) after large scale purification. These protein fractions were further purified by CL-Sepharose 6B column chromatography which resolved fractions from CP4 that recognized the ER bound by estrogen only or antiestrogen only. Thus, these hydrophobic chromosomal proteins enhanced the binding of the ER to reconstituted chromatin. To further investigate the interaction of ERBFs with ER, gel mobility shift assays were performed. The highly purified CP4 fraction with ERBF activity in the binding assay with reconstituted chromatin caused an increase in the formation of the retarded ER-estrogen responsive element (ERE) band. Thus, chromatin contains specific ERBFs for ER bound by estrogen which enhance the binding of ER to genomic DNA and a target ERE sequence. Further purification of the CL-Sepharose fraction with ERBF activity was achieved by preparative SDS-PAGE. ERBF activity was attributed to proteins with approximate molecular weights of 16,000, 13,000, and 12,000 and a pl of > 9.0. Peptides were partially sequenced by Edman degradation and were found to have identity with histones H2B and H4. A 17 kDa protein without ERBF activity was identified as H3. Since these histones were not readily extracted from chromatin with 3 M NaCl or 1-3 M GdnHCl, we postulate that some ERBFs may be histone variants or modified histones that display a very high affinity for DNA and ER.