Soy protein isolate, containing genistein, daidzein and glycitein, was used to study the profiles of soy isoflavone metabolites in Sprague-Dawley rats. After 50 days, rat liver tissues, serum and urine were analyzed by HPLC-MS and HPLC-ECD. HPLC-APCI-MS results showed that daidzein, equol, genistein and O-DMA were detected. Interestingly, the aglycone and the conjugated forms of formononetin, which is a methylated derivative of daidzein found in many plants, were also observed in rat liver and serum, but not in urine. The respective concentrations of these two forms of formononetin were 0.056 ± 0.022 and 0.621 ± 0.116 nmoL/g (mean ± SD) in liver tissue, and were 0.005 ± 0.001 and 0.035 ± 0.002 nmoL/mL in rat serum. Formononetin in conjugated forms were 90.8% in liver tissue and 85.7% in serum by calculation. Incubation of daidzein with liver homogenate, microsomes, and catechol 4’-O-methyl transferase (COMT) resulted in a 7~10% conversion of daidzein to the aglycone form of formononetin. Formononetin is proposed to be a metabolite of O-methylation of daidzein in rat liver and exists mainly in conjugated forms.