Estrogen-dependent transcriptional activation by estrogen receptor alpha (ERalpha) depends on the conformation of helices 3 and 12 in the ligand-binding domain. To better understand the function of helix 3 in ERalpha, we examined the role of charged residues, which are conserved in most steroid receptors in helix 3, in estrogen-dependent transactivation. The replacement of Asp-351 with lysine (D351K) or leucine (D351 L) completely abolished estrogen-dependent transactivation without affecting estrogen-binding, DNA-binding and homodimerization activities in ERalpha. The mutations dramatically reduced the ligand-dependent activation function 2 activity and impaired the ability of ERalpha to bind p160 coactivators. In addition, the D351K mutant effectively inhibited the transcriptional activation activity of wild-type ERalpha. Furthermore Asp-351 was required not only for the estrogen-dependent conformational change of wild-type ERalpha but also for the constitutive transcriptional activity and ligand-independent active conformation of ERalpha mutant Y537N. Similarly, in the orphan nuclear receptor called estrogen-related receptor 3 (ERR3), the replacement of Asp-273 (the corresponding amino acid to Asp-351 in ERalpha) with lysine abolished constitutive transcriptional activity of ERR3 without affecting DNA-binding activity and impaired the ability of the receptor to interact with p160 coactivators. These data suggest a role of Asp-351 in inducing and stabilizing the active conformation of ERalpha, and our results experimentally confirm the concept that Asp-351 in helix 3 interacts with the amide hydrogen of L540 in helix 12 to form a transcriptionally competent surface for binding p160 coactivators.