We have studied the uptake of [3H]-medicarpin and its glutathione conjugate(s) into vacuolar membrane vesicles from etiolated hypocotyls of mung bean (Vigna radiata). Unconjugated medicarpin is taken up at a low rate in the presence or absence of MgATP. However, [3H]-medicarpin-glutathione conjugate(s), prepared by incubation of medicarpin with a total maize glutathione S-transferase preparation, is taken up more than four-fold faster than medicarpin in the presence of MgATP, and this uptake is MgATP-dependent. Uptake of medicarpin-glutathione was not significantly inhibited by the ionophore gramicidin-D, but was strongly inhibited by vanadate and the alternative transport substrate S-(2,4-dinitrophenyl) glutathione. Our results demonstrate, in a model system, the potential utilization of the high affinity, high capacity, uncoupler-insensitive glutathione conjugate pump for the vacuolar transport of an isoflavonoid phytoalexin.