Key residues of the human mineralocorticoid receptor (hMR) involved in the recognition of agonist and antagonist ligands were identified by alanine-scanning mutagenesis based on a homology model of the hMR ligand-binding domain. They were tested for their transactivation capacity and ability to bind agonists (aldosterone, cortisol) and antagonists (progesterone, RU26752). The three-dimensional model reveals two polar sites located at the extremities of the elongated hydrophobic ligand-binding pocket. Mutations of Gln776 and Arg817 in site I reduce the affinity of hMR for both agonists and antagonists and affect the capacity of hMR to activate transcription, suggesting that the C3-ketone group, common to all ligands, is anchored by these two residues conserved within the nuclear steroid receptor family. In contrast, mutations of Asn770 and Thr945 in the opposite site only affect the binding of agonists bearing the C21-hydroxyl group. The binding of hMR antagonists that exhibit a smaller size and faster off-rate kinetics compared with agonists is not affected. In the light of the hMR homology model, a new mechanism of antagonism is proposed in which the AF2-AD core region is destabilized by the loss of contacts between the antagonist and the helix H12 region.