The mineralocorticoid receptor (MR) plays a central role in electrolyte homeostasis and in cardiovascular disease. We have previously reported a ligand-dependent N/C-interaction in the MR. In the present study we sought to fully characterise the MR N/C-interaction. By utilising a range of natural and synthetic MR ligands in a mammalian two-hybrid assay (M-2-H) we demonstrate that in contrast to aldosterone, which strongly induces the interaction, the physiological ligands deoxycorticosterone (DOC) and cortisol weakly promote the interaction but predominantly inhibit the aldosterone mediated N/C-interaction. Similarly, progesterone and dexamethasone antagonise the interaction. In contrast, the synthetic agonist 9-fludrocortisol robustly induces the interaction. The ability of the N/C-interaction to discriminate between MR agonists suggests a subtle conformational difference in the ligand-binding domain induced by these agonists. We also demonstrate that the N/C-interaction is not cell-specific, consistent with the evidence from a GST pull-down assay, of a direct protein-protein interaction between the N- and C-terminal domains of the MR. Examination of a panel of deletions in the N-terminus suggests that several regions may be critical to the N/C-interaction. These studies have identified functional differences between physiological MR ligands which suggest that the ligand-specific dependence of the N/C-interaction may contribute to the differential activation of the MR which has been reported in vivo.