The synthetic progestins, medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-EN or NET-A), are widely used as female contraceptive agents and in hormone replacement therapy (HRT). Competitive binding revealed that MPA displays a higher relative binding affinity than NET-A and progesterone (prog) for the human GR (Kd of 4.2 nM for dexamethasone (dex) and Ki's of 10.8, 270 and 215 nM for MPA, NET-A and prog, respectively). Furthermore, MPA displays much greater glucocorticoid (GC) transactivation agonist potency than NET-A or prog (EC50s of 1.1, 7.2, >1000 and 280 nM for dex, MPA, NET-A and prog, respectively) and much greater GC agonist potency for transrepression than NET-A or prog (EC50s of 0.21, 2.7, >100 and 26 nM for dex, MPA, NET-A and prog, respectively). In addition, MPA induces phosphorylation of the GR at Ser 211 to a much greater extent than NET-A or prog and protects the GR from partial trypsin digestion in vitro to a much greater extent than NET-A or prog at saturating concentrations. Together these results suggest that the differences in biological activity of the progestins are not merely due to differences in their affinity for the GR but also due to the induction of different conformational changes in the liganded-GR. MPA and NET-A therefore display very different GC-like properties compared to each other and to prog, and are likely to exhibit different side effects via the GR.