Incubation of aldosterone with confluent layers of A6 (toad kidney) cells leads to its hydroxylation at the 6 beta-position. 6 beta-Hydroxyaldosterone is the major metabolite when the incubation is carried out at pH 6.8, whereas the product comprises 6 beta-hydroxy-17-isoaldosterone accompanied by some 6 beta-hydroxyapoaldosterone at pH 7.4. All products were identified by high-field 1H nuclear magnetic resonance spectroscopy. Control experiments indicated that the side-chain isomerization to form the 17-iso and apo derivatives occurs after the cytochrome P 450-dependent synthesis of 6 beta-hydroxyaldosterone.