The isoflavones of the roots of Pueraria lobata (Willd.) Ohwi (Puerariae Radix) were investigated by high-performance liquid chromatography (HPLC) coupled to photodiode array (PDA) and to mass spectroscopy (MS) using atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) in combination with collision-activated decomposition (CAD) (HPLC-APCI-CAD-MS or ESI-CAD-MS) for identification of glycosides and HPLC-APCI-CAD-MS for identification of aglycones. The major glycosides are derived from daidzein (9) and most are 8-C-glycosides. 3'-Hydroxypuerarin-4'-O-deoxyhexoside (2b) and 3'-methoxy-6 "-O-D-xylosylpuerarin (6) were identified as new constituents. MS data were obtained for puerarin-4'-O-D-glucoside (1), 3'-hydroxypuerarin (2a), puerarin (3), 3'-methoxypuerarin (4), 6 "-O-D-xylosylpuerarin (5), daidzin (7) and 3'-methoxydaidzin (8), which were previously characterized by NMR analysis. Isoflavones identified in Puerariae Radix comprise 3'-methoxydaidzein (10), genistein (12), daidzein-7-O-methyl ether (13a), 3'-methoxydaidzein-7-O-methyl ether or 3'-methoxyformononetin (13b) and biochanin A (15), while previous characterization of daidzein (9) and formononetin (14) was substantiated by MS data. The structure of compound 11 could not be established by MS techniques. The estrogenic activity was mainly located in the aglycone fraction.