Isoflavones in the methanolic extracts of kudzu (Pueraria lobata) callus, suspension and root cultures were compared in order to develop an experimental system in which puerarin (daidzein 8-C-glucoside) and other isoflavones could be synthesised in vitro. Quantitative variation of puerarin and other known isoflavones was estimated in kudzu culture extracts using HPLC-UV. The highest and lowest amounts of puerarin (14.56 and 0.33 mg/g) were present in in vitro root cultures and leaf tissue-derived callus cultures, respectively. A total of 48 isoflavone metabolites were detected in extracts of kudzu root cultures by HPLC-MS/MS, and the structures of 33 of them were tentatively assigned. Amongst these, 12 isoflavone C-glycosides were identified. Hydroxyderivatives of puerarin in several isomeric forms were detected, some of which have not been previously reported in kudzu root. The molecular weights, interpretation of characteristic fragment ions obtained from HPLC-MS/MS and comparison with reported data allowed the putative identification of the isoflavone metabolites.