The knots of Wisteria brachybotrys Sieb. et Zucc. (Leguminosae) have been used in Japanese folk medicine for the treatment of gastric cancer. In a previous paper, some triterpenes and triterpenoid saponins were reported to show significant inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation. As part of a continuing search for novel, naturally occurring potential antitumor promoters from medicinal plants, the MeOH extract of knots of W. brachybotrys was found to have significant inhibitory effects on EBV-EA activation in Raji cells. Many compounds that inhibit EBV-EA induction by tumor promoters act as inhibitors of tumor promotion in vivo. Furthermore, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances 32P incorporation into cell phospholipids and plays an important role in the early stages of tumor promotion in vivo. Therefore, the inhibitory effects of isoflavonoids from W. brachybotrys on EBV-EA activation and TPA-stimulated 32P incorporation into phospholipids of HeLa cells were examined. Bioassay-directed fractionation of the active extract led to the isolation and characterization of afromosin [1], formononetin [2], wistin [3], and ononin [4] as inhibitory principles on EBV-EA activation. To investigate the inhibitory mechanism, some of these isoflavonoids were also tested for their effect on binding to the TPA receptor in an epidermal particulate fraction using the cold Me₂CO filter method. Although these isoflavonoids were previously isolated from Wisteria floribunda, their potent antitumor promoter activities are reported here for the first time.