We have confirmed that A6 cells (derived from kidney of Xenopus laevis), which contain both mineralocorticoid and glucocorticoid receptors, do not normally possess 11-hydroxysteroid dehydroxgenase (11-HSD1 or 11-HSD2) enzymatic activity and so are without apparent "protective" enzymes. A6 cells do not convert the glucocorticoid corticosterone to 11-dehydrocorticosterone but do, however, possess steroid 6-hydroxylase that transforms corticosterone to 6-hydroxycorticosterone. This hydroxylase is cytochrome P-450 3A (CYP3A). We have now determined the effects of 3,5-tetrahydroprogesterone and chenodeoxycholic acid (both inhibitors of 11-HSD1) and 11-dehydrocorticosterone and 11-hydroxy-3,5-tetrahydroprogesterone (inhibitors of 11-HSD2) and carbenoxalone, which inhibits both 11-HSD1 and 11-HSD2, on the actions and metabolism of corticosterone and active Na⁺ transport [short-circuit current (I_sc)] in A6 cells. All of these 11-HSD inhibitory substances induced a significant increment in corticosterone-induced I_sc, which was detectable within 2 h. However, none of these agents caused an increase in I_sc when incubated by themselves with A6 cells. In all cases, the additional I_sc was inhibited by the mineralocorticoid receptor (MR) antagonist, RU-28318, whereas the original I_sc elicited by corticosterone alone was inhibited by the glucocorticoid receptor antagonist, RU-38486. In separate experiments, each agent was shown to significantly inhibit metabolism of corticosterone to 6-hydroxycorticosterone in A6 cells, and a linear relationship existed between 6-hydroxylase inhibition and the MR-mediated increase in I_sc in the one inhibitor tested. Troleandomycin, a selective inhibitor of CYP3A, inhibited 6-hydroxylase and also significantly enhanced corticosterone-induced I_sc at 2 h. These experiments indicate that the enhanced MR-mediated I_sc in A6 cells may be related to inhibition of 6-hydroxylase activity in these cells and that this 6-hydroxylase (CYP3A) may be protecting the expression of corticosterone-induced active Na⁺ transport in A6 cells by MR-mediated mechanism(s).