Recent studies have shown that the mammalian lignans enterodiol (END) and enterolactone (ENL) are biotransformed in vitro by hepatic microsomes from rats and humans to various metabolites carrying one additional hydroxy group either at the aromatic or at the aliphatic moiety. To clarify whether these metabolites are also formed in vivo, each lignan was administered intraduodenally at a dose of 10 mg/kg of bw to bile duct-catheterized female Wistar rats and the 6 h bile analyzed by HPLC and GC-MS. With END-dosed rats, three products of aromatic and two of aliphatic monohydroxylation were found, whereas six aromatic and five aliphatic monohydroxylated biliary metabolites were detected after administration of ENL. The metabolites hydroxylated at the aromatic rings were unequivocally identified by comparison with synthetic reference compounds. The structures of the in vivo metabolites arising from aliphatic hydroxylation could not be completely elucidated; they were identical with some of the formerly reported microsomal products according to GC retention times and mass spectra. Significant amounts of most of the metabolites of the mammalian lignans identified in bile were also found in the urine of female rats after oral administration of 10 mg/kg of bw END or ENL and in the urine of female and male Wistar rats after they had been fed a diet containing 5% flaxseed. Thus, the mammalian lignans END and ENL give rise to several hydroxylated metabolites in vivo, which may contribute to the biological effects of these important food constituents.