The existence of two rather than one estrogen receptor, today characterized as estrogen receptor α (ERα) and estrogen receptor β (ERβ), indicates that the mechanism of action of 17β-estradiol and related synthetic drugs is more complex than previously thought. Because the homology of amino acid residues in the ligand-binding domain (LBD) of ERβ is high compared with those amino acid residues in ERα LBD, previously shown to line the ligand binding cavity or to make direct contacts with ligands, it is not surprising that many ligands have a similar affinity for both receptor subtypes. We report that 17α-ethynyl,17β-estradiol, for example, has an ERα-selective agonist potency and that 16β,17α-epiestriol has an ERβ-selective agonist potency. We also report that genistein has an ERβ-selective affinity and potency but an ERα-selective efficacy. Furthermore, we show that tamoxifen, 4-OH-tamoxifen, raloxifene, and ICI 164,384 have an ERα-selective partial agonist/antagonist function but a pure antagonist effect through ERβ. In addition, raloxifene displayed an ERα-selective antagonist potency, in agreement with its ERα-selective affinity. However, although ICI 164,384 showed an ERβ-selective affinity, it had a similar potency to antagonize the effect of 17β-estradiol in the ERα- and ERβ-specific reporter cell lines, respectively. In conclusion, our data indicate that the ligand binding cavity of ERβ is probably more different from that of ERα than can be anticipated from the primary sequences of the two ER subtypes and that it will be possible to develop receptor-specific ligands that may form the basis of novel pharmaceuticals with betterin vivo efficacy and side effect profile than current available drugs.