Legume plants have an ability to fix atmospheric nitrogen into nutrients via symbiosis with soil microbes. As the initial event of the symbiosis, legume plants secrete flavonoids into the rhizosphere to attract rhizobia. Secretion of flavonoids is indispensable for the establishment of symbiotic nitrogen fixation, but almost nothing is known about the membrane transport mechanism of flavonoid secretion from legume root cells. In this study, we performed biochemical analyses to characterize the transport mechanism of flavonoid secretion using soybean (Glycine max) in which genistein is a signal flavonoid. Plasma membrane vesicles prepared from soybean roots showed clear transport activity of genistein in an ATP-dependent manner. This transport activity was inhibited by sodium orthovanadate, a typical inhibitor of ATP-binding cassette (ABC) transporters, but was hardly affected by various ionophores, such as gramicidin D, nigericin, or valinomycin, suggesting involvement of an ABC transporter in the secretion of flavonoids from soybean roots. The K(m) and V(max) values of this transport were calculated to be 158 mum and 322 pmol mg protein(-1) min(-1), respectively. Competition experiments using various flavonoids of both aglycone and glucoside varieties suggested that this ABC-type transporter recognizes genistein and daidzein, another signaling compound in soybean root exudates, as well as other isoflavonoid aglycones as its substrates. Transport activity was constitutive regardless of the availability of nitrogen nutrition. This is, to our knowledge, the first biochemical characterization of the membrane transport of flavonoid secretion from roots.