A6 cells form typical tight epithelia when grown in culture on permeable supports and exhibit active Na+ transport [short circuit current (Isc)], which is stimulated by aldosterone and corticosterone. Previous studies demonstrated nuclear binding of polar corticosterone metabolites produced by the cells. This study was performed to determine whether sufficient quantities of the metabolite(s) are released into the medium of A6 cells for identification and to test for agonist activity on active Na+ transport. Cells were incubated in [3H]corticosterone (10(-8)-10(-4) M) for 24 h. Approximately 25-35% of the radiolabel, recovered in ethyl acetate extracts of medium, chromatographed on reverse phase HPLC as a single peak more polar than corticosterone. This derivative cochromatographed with 6 beta-hydroxycorticosterone (6 beta-OH-corticosterone) on HPLC and normal phase high performance TLC. Mass spectroscopy of 6 beta-OH-corticosterone and the unknown yielded 10 identical molecular ions, including the molecular ion with a mass to charge ratio of 362 corresponding to the mol wt of 6 beta-OH-corticosterone stimulated Isc in A6 epithelia with a time course typical of a steroid and an EC50 of 10(-6) M. The Isc induced by 6 beta-OH-corticosterone was equivalent to net Na+ flux, indicating active Na+ transport stimulation. At maximum effective concentrations of corticosteroids, 6 beta-OH-corticosterone plus aldosterone induced a greater Isc stimulation than aldosterone alone, suggesting that at least a portion of the effect of 6 beta-OH-corticosterone is mediated by a steroidal pathway other than that used by aldosterone. Also, corticosterone produced twice the Isc increase produced by aldosterone. Thus, 6 beta-OH-corticosterone may contribute to the enhanced corticosterone effect on Isc compared to aldosterone alone.