Gel shift assays were employed to distinguish between the contribution of 17 beta-estradiol (E2) and a short heating step to the ability of the rat uterine cytosolic estrogen receptor (ER) to bind to the estrogen response element (ERE) from the vitellogenin A2 gene (vitERE). Despite the popularity of models in which the ER is a ligand-activated DNA-binding protein, these studies find that estrogen does not significantly contribute to receptor-DNA complex formation. An avidin-biotin complex with DNA (ABCD) assay was utilized to obtain quantitative measurement of the affinities of the ER for the vitERE and a mutant sequence. Scatchard analysis gave a dissociation constant of 390 +/- 40 pM for the E2-occupied, heated ER to the vitERE. The data fit a one-site model and evidence for cooperatively was not observed. A dissociation constant of 450 +/- 170 pM was obtained for the unoccupied, heated ER, leading to the conclusion that estrogen was not necessary for specific binding to DNA. The percentage of ER capable of binding vitERE varied with each cytosol preparation, ranging from 60 to 100% and estrogen did not appear to affect this variation. Competition against the vitERE with a 2-bp mutant sequence showed a 250-fold lower relative binding affinity of the receptor for the mutant over the vitERE sequence. This ability of the ER to discriminate between target and nonspecific DNA sequences was also not dependent on the presence of estrogen.