The ligand-binding domains of steroid hormone receptors possess a conserved structure with 12 alpha-helices surrounding a central hydrophobic core. On agonist binding, a repositioned helix 12 forms a pocket with helix 3 (H3) and helix 5 (H5), where transcriptional coactivators bind. The precise molecular interactions responsible for activation of these receptors remain to be elucidated. We previously identified a H3-H5 interaction that permits progesterone-mediated activation of a mutant mineralocorticoid receptor. We were intrigued to note that the potential for such interaction is widely conserved in the nuclear receptor family, indicating a possible functional significance. Here, we demonstrate via transcriptional activation studies in cell culture that alteration of residues involved in H3-H5 interaction consistently produces a gain of function in steroid hormone receptors. These data suggest that H3-H5 interaction may function as a molecular switch regulating the activity of nuclear receptors and suggest this site as a general target for pharmacologic intervention. Furthermore, they reveal a general mechanism for the creation of nuclear receptors bearing increased activity, providing a potentially powerful tool for the study of physiologic pathways in vivo.