Metabolism of glucocorticoids to A-ring-reduced dihydro- and tetrahydro-derivatives by means of hepatic 5alpha- and 5beta-reductases has long been regarded as a pathway of irreversible inactivation. However, 5alpha-reduced metabolites of other steroids, e.g. testosterone and aldosterone, have significant biological activity. We investigated whether 5alpha-reduced metabolites of corticosterone are glucocorticoid receptor (GR) agonists. Corticosterone, 5alpha-tetrahydrocorticosterone (5alphaTHB), and 5alpha-dihydrocorticosterone (5alphaDHB) were similarly effective in displacing tritiated dexamethasone from binding sites in hepatocytes, whereas 5beta-reduced metabolites were less effective in binding. 5alphaTHB had glucocorticoid receptor agonist effects in vitro and in vivo. After transient co-transfection of hGR and a murine mammary tumor virus-luciferase reporter into HeLa cells, 5alphaTHB was active to a comparable extent as corticosterone (28-fold versus 37-fold induction, respectively, at 1 microm) and additive to the effect of corticosterone. 5beta-Reduced metabolites did not activate GR. In H4IIE hepatoma cells, both 5alphaTHB and corticosterone induced mRNA expression of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase (6.0-versus 10.1-fold and 3.5-versus 3.9-fold at 1 microM, respectively), an effect that was inhibited by RU486. To assess in vivo glucocorticoid activity, suppression of plasma ACTH was demonstrated in adrenalectomized rats after intraperitoneal administration of vehicle (ACTH trough 80.2 pm), corticosterone (5 mg/kg; 22 pm, p <0.001) or 5alphaTHB (5 mg/kg; 51.3 pm, p <0.005). Similar endogenous concentrations of corticosterone and 5alphaTHB were detected in rat liver homogenates by gas chromatography mass spectrometry. We conclude that 5alpha-reduced glucocorticoids bind to and activate GR. Transcription of glucocorticoid-regulated genes in tissues that express 5alpha-reductases will thus be influenced by intracellular levels of both corticosterone and its 5alpha-reduced metabolites.