DNA-cellulose competition binding assays were used to measure the ability of cloned DNA fragments of the chicken vitellogenin II gene to displace the estrogen-receptor complex from total chicken DNA coupled to cellulose. The DNA fragment that gave the highest competition is situated in the upstream region of the gene between nucleotides -458 and -725. This DNA fragment has four small clusters of A + T-rich sequences and contains the estrogen-dependent hypomethylation site. In vitro methylation of the Msp I site does not change the capacity of the DNA fragment to compete for estrogen-receptor complex, whereas cleavage of the C-C-G-G (Msp I site) results in a complete loss of competition of this fragment for estrogen-receptor complex. These results, combined with deoxyribonuclease I protection experiments, suggest that the most probable binding site for estrogen-receptor complex is . . .G-C-G-T-G-A-C-C-G-G-A-G-C-T-G-A-A-A-G-A-A-C-A-C. . . . This sequence has 73% homology with the core enhancer sequence of simian virus 40, . . .G-G-T-G-T-G-G-A-A-A-G. . . (identical bases italicized).