The oxidative metabolism of the major soy isoflavones daidzein and genistein was investigated using liver microsomes from Aroclor-treated male Wistar rats. Both daidzein and genistein were extensively metabolized and are therefore excellent substrates for cytochrome P450 enzymes. The identity of the metabolites was elucidated using high-performance liquid chromatography (HPLC) with diode array detection, gas chromatography-mass spectrometry (GC/MS), and HPLC/atmospheric pressure ionization electrospray mass spectrometry (API-ES MS) as well as reference substances. Daidzein was converted to nine metabolites, comprising four monohydroxylated, four dihydroxylated, and one trihydroxylated metabolite. Genistein was metabolized to four monohydroxylated and two dihydroxylated products. With both isoflavones the additional hydroxy groups are exclusively introduced into the ortho positions of existing phenolic hydroxy groups. The major metabolites of daidzein were identified as 6,7,4'-trihydroxyisoflavone, 6,7,3',4'-tetrahydroxyisoflavone, 7,8, 4'-trihydroxyisoflavone, and 5,6,7,4'-tetrahydroxyisoflavone. The main microsomal metabolites of genistein were 5,6,7, 4'-tetrahydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone. Furthermore, the GC/MS and HPLC/API-ES MS analysis support the conclusion that one monohydroxylated metabolite of daidzein and genistein is hydroxylated at the aliphatic position C-2 of the C-ring. The UV-vis, GC/MS, and HPLC/MS data of all detected metabolites as well as the derived chemical structure of the metabolites are presented. Most metabolites are reported in this paper for the first time. On the basis of these findings it is suggested that hydroxylation reactions may also play an important role in the in vivo metabolism of the soy isoflavones daidzein and genistein.