Puerarin (daidzein-8-C-glucoside) is the major bioactive isoflavone of kudzu root (the root of Pueraria lobata). Its metabolic fate, however, is not well-known. In this study, a sensitive and specific LC-ESI-MS/MS method for the determination of puerarin and its metabolites daidzein, dihydrodaidzein, and equol was developed for their analysis in biological samples. Two new metabolites of puerarin, mono- and dihydroxylated derivatives, were detected in the urine and feces of rats after oral administration. The persistence of puerarin in blood and urine as the principal metabolic form for the period of 4-72 h after oral administration suggested that puerarin is rapidly absorbed from the intestine without metabolism. Its presence in organs such as the brain suggests that this glucoside may enter tissues by specific transport pathways. Study of these metabolites may provide further understanding of the health beneficial effects of puerarin in kudzu dietary supplements.