Soy isoflavones have recently gained considerable interest due to their possible health benefits. However, detailed studies on the metabolism of isoflavones are lacking. The aims of the investigation presented here were (1) to study the in vitro intestinal metabolism of isoflavones and their hydroxylated analogues 3'-OH-daidzein, 6-OH-daidzein, 8-OH-daidzein, and 3'-OH-genistein and (2) to characterize the structures of some earlier identified urinary metabolites of soy isoflavones, for which no authentic reference compounds have been available. Isoflavone standards (1-2 mg) were fermented with human fecal flora (16.7%) for 24 h. Metabolites formed during the fermentation were tentatively identified by interpretation of the mass spectra of trimethylsilylated compounds obtained by GC-MS. Compounds having hydroxyl groups at 5-position (i.e., genistein and 3'-OH-genistein) were completely converted to metabolites that could not be detected by the methods used in this study. The metabolism of daidzein and its hydroxylated analogues, 3'-OH-daidzein, 6-OH-daidzein, and 8-OH-daidzein, occurred to a much lesser extent. Minor amounts of reduced metabolites (i.e., isoflavanones and -methyldeoxybenzoins) of these compounds were tentatively identified in fermentation extracts. The retention times and the mass spectra of reduced isoflavone metabolites, obtained from in vitro fermentations of pure compounds, were utilized to identify unknown urinary metabolites of soy isoflavones. Four novel isoflavone metabolites were identified in human urine collected after soy supplementation: 3' '-OH-O-desmethylangolensin, 3',4',7-trihydroxyisoflavanone, 4',7,8-trihydroxyisoflavanone, and 4',6,7-trihydroxyisoflavanone.