The most important determinants of glucocorticoid (GC) and mineralocorticoid (MC) action in target tissues are the prereceptor metabolism by 11β-hydroxysteroid-dehydrogenases (HSDs) and receptor transactivation. We characterized these parameters for a wide range of steroids often used in clinical practice. The 11-hydroxy-group is essential to GC and MC receptor transactivation. As HSD2 oxidizes 11-hydroxysteroids such as cortisol and prednisolone to inactive 11-ketosteroids such as cortisone and prednisone, GC and MC activity in HSD2-expressing tissues (kidney, colon, placentofetal unit) is strongly regulated by this enzyme. As 6α or 9α fluorination (such as in 9α-fluorcortisol) decreases HSD2 oxidation and increases both GC and MC receptor transactivation, these modifications lead to optimal, but unselective MC and GC receptor occupation. Increased GC and decreased MC receptor transactivation leading to more selective GC activity is reached by the following substituents: 16-methylene (in prednylidene), 16, 17-methyloxazoline (in deflazacort), 16α-methyl (in dexamethasone), 16β-methyl (in betamethasone) and Δ1-dehydroconfiguration (in prednisolone). While modifications at position 16 decrease HSD2 oxidation, the Δ1-dehydroconfiguration increases HSD2 activity leading to an enhanced inactivation of prednisolone compared to all other steroids tested. Our in vitro data allow us to conclude that 9α-fluorcortisol, the most frequently used substance for MC substitution, seems the best choice of available steroids for this approach. Whereas GC selectivity can be improved by hydrophobic substituents at position 16, such as prednylidene and deflazacort, and the Δ1-dehydroconfiguration, maximal GC activity needs additional fluorination at position 6α and/or 9α such as dexamethasone. For GC therapy directed to HSD2-expressing organs, widely used prednisolone does not seem to be the optimal recommendation.