A high-performance liquid chromatographic method was developed for the determination of ipriflavone and its metabolites, M1 and M5, in rat plasma, urine, and tissue homogenate using an internal standard, testosterone for ipriflavone or diazepam for M1 and M5. The method involved deproteinization followed by injection onto a C18 reversed-phase column. The mobile phases were 0.05 M acetate buffer (pH 3):acetonitrile:methanol (40:35:25, v/v/v) for ipriflavone and 0.05 M acetate buffer (pH 3):acetonitrile:methanol:phosphoric acid (50:25:25:0.1, v/v/v/v) for M1 and M5, and run at a flow rate of 1.5 ml/min. The column effluent was monitored by a UV detector set at 254 nm. The retention times for testosterone, ipriflavone, M1, M5, and diazepam were approximately 6, 12, 6, 8, and 10 min, respectively. The detection limits for I, M1, and M5 in rat plasma were 20, 20, and 50 ng/ml, respectively, and the corresponding values in rat urine and tissue homogenate were 50-100 ng/ml. The coefficients of variation of the assay were generally low (below 9.84%) for rat plasma, urine, and tissue homogenate. No interferences from endogenous substances were found.