Affinity of the mineralocorticoid receptor (MR) is similar for aldosterone and the glucocorticoids (GC) cortisol and corticosterone, which circulate at concentrations far exceeding those of aldosterone. 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) inactivation of GC within the immediate vicinity of the MR is credited with prereceptor specificity for aldosterone in cells coexpressing MR and 11βHSD2. 11βHSD2 efficacy is also critical to other recently described 11βHSD2 substrates. The aim of this work was to address doubts that low levels of expression of 11βHSD2 in aldosterone target tissues suffice to prevent the initiation of gene transcription by the MR activated by physiological concentrations of corticosterone. Cell models stably expressing an MR/ Gaussia luciferase reporter and various levels of constitutive or induced 11βHSD2 at concentrations lower than those in rat kidney homogenates and microsomes were produced. Aldosterone and corticosterone were equipotent transactivators of the MR reporter gene in cells without 11βHSD2. Rate of conversion of tritiated corticosterone to 11-dehydrocorticosterone increased and corticosterone-induced nuclear translocation of MR decreased, as 11βHSD2 expression increased. The 50% maximal MR activation for the reporter gene stimulation by corticosterone rose with increasing 11βHSD2 expression, shifting the steroid dose-response curve for corticosterone-induced MR transactivation to the right. Several stable cell lines expressing an easily and reproducibly measured MR reporter system and consistent incremental amounts of 11βHSD2 protein were produced and used to document that 11βHSD2 within low physiological levels inactivates relevant concentrations of GC and decreases MR transactivation by GC in a dose-dependent fashion, laying to rest doubts of the efficacy of this enzyme.