When roots of 5-d-old red clover (Trifolium pratense L.) seedlings were treated with chitohexaose and CuCl(2), constitutive glucosidic conjugates of formononetin (F) and (-)-maackiain (Ma) promptly disappeared. Free F and Ma, which were not detected in the control tissues, rapidly appeared to reach the maximum levels 24 h after the initiation of treatment and then declined. The pattern of appearance and disappearance was the same between the tissues treated with chitohexaose and CuCl(2). The enzyme activities related to isoflavonoid metabolism were investigated using crude extracts from elicitor-treated roots. The conjugate-forming glucosyltransferase and malonyltransferase activities were lost or markedly reduced after elicitor treatment. On the other hand, malonylesterase and glucosidase activities remained unchanged or showed only slight increase. Phenylalanine ammonia-lyase activity disappeared following elicitor treatment. These results indicated that free aglycones were produced from the conjugate pool by hydrolysis under conditions in which the biosynthetic pathway was extinguished. The amount of Ma produced did not explain that of MaGM lost (about 45%). Since Ma, but not its conjugates, served as a substrate for peroxidase from the elicitor-treated roots, Ma was considered to be converted to insoluble materials.