Microsomal monoxygenases can oxidize the axial hydroxyl of the terminal digitoxosyl of digoxin (dg-3), digoxigenin bis-, and digoxigenin mono-digitoxoside (dg-2 and dg-1, respectively) to an oxo-group. The corresponding metabolites (15'-dehydro-dg-3, 9'-dehydro-dg-2, and 3'-dehydro-1, respectively) have been identified by chromatographic and chemical methods. Only after this oxibation the terminal sugar can be split off, presumably by beta-elimination. Therefore, for the degradation of dg-3 three successive cytochrome P450 catalyzed oxidations are necessary before digoxigenin (dg-0) can be obtained. The highest oxibation rate was observed with dg-1 (120-150 pmoles/mg microsomal protein/min) and by far the lowest with dg-2 (6-7 pmoles/min) as the substrate (concentration was 30/microM). The latter may contribute to the effect that dg-2 is the main dg-3 metabolite in vivo. Pretreatment of rats with canrenoate enhanced the microsomal oxidation of dg-3, dg-2, and dg-1 by a factor of 3.2, 2.3 and 1.3, respectively. In contrast, there was no increase after pretreatment with phenobarbital.