Metabolites of [G-3H]digitoxin in the rat bile have been studied by combining column chromatography on Sephadex LH 20 and TLC on silica gel. Bile was collected during 6 hrs, after i.p. injection of 800 mug/kg of [G-3H]digitoxin. The following digitoxigenin containing compounds were separated: -mono-digitoxoside (43%, mostly conjugated), -bis-digitoxoside and digitoxin (12% and 2% resp., mainly non-conjugated). C-12beta-hydroxylated metabolites related to digoxigenin were identified as -bis-digitoxoside (18%), digoxin (4%) and -mono-digitoxoside (traces only). The results indicate that even with regard to the C12-hydroxylation the main metabolic pathway of digitoxin proved to be the cleavage of the third and second digitoxose and the conjugation of digitoxigenin -mono-digitoxoside. However the cleavage of the genin linked digitoxose as well as the epimerisation of the genin do not play a role in the detoxication of digitoxin in vivo. Besides these findings three groups of yet unknown metabolites amounting up to 10% of the excreted radioactivity were separated from the identified metabolites. Acid hydrolysis of these compounds yielded digitoxigenin or digoxigenin. Therefore it may be concluded that metabolic changes in the sugar moiety are their common characteristics.