The present study demonstrates that the nature of the binding of estrogens to the hormone-binding domain of the estrogen receptor (ER) modifies the responses of estrogen-dependent cells. We report here that 10 nM estradiol (E2) forms noncovalent associations with the ER, increases the level of ER and Progesterone Receptors (PR) in ER+ MCF-7 human breast cancer cells in culture following short-term or long-term exposure to E2. In contrast, 10 nM 16-alpha-hydroxyestrone (16 alpha-OHE1), a physiological metabolite of E2, in short-term cultures is equivalent to E2, but upon long-term incubation, 16 alpha-OHE1 forms covalent associations with the ER, produces a marked decrease in ER and PR levels reaching values similar to, or below to that of control cells.