In this paper we present results on the vacuolar uptake mechanism for two flavone glucuronides present in rye mesophyll vacuoles. In contrast to barley flavone glucosides (Klein et al. (1996) J. Biol. Chem. 271, 29666-29671), the flavones luteolin 7-O-diglucuronyl-4'-O-glucuronide (R1) and luteolin 7-O-diglucuronide (R2) were taken up into vacuoles isolated from rye via a directly energized mechanism. Kinetic studies suggested that the vacuolar glucuronide transport system is constitutively expressed throughout rye primary leaf development. Competition experiments argued for the existence of a plant MRP-like transporter for plant-specific and non-plant glucuronides such as beta-estradiol 17-(beta-D-glucuronide) (E217G). The interaction of ATP-dependent vacuolar glucuronide uptake with glutathione and its conjugates turned out to be complex: R1 transport was stimulated by dinitrobenzene-GS and reduced glutathione but was inhibited by oxidized glutathione in a concentration-dependent manner. In contrast, R2 uptake was not increased in the presence of reduced glutathione. Thus, the transport system for plant-derived glucuronides differed from the characteristic stimulation of vacuolar E217G uptake by glutathione conjugates but not by reduced glutathione (Klein et al. (1998) J. Biol. Chem. 273, 262-270). Using tonoplast vesicles isolated with an artificial K+ gradient, we demonstrate for the first time for plant MRPs that the ATP-dependent uptake of R1 is membrane-potential dependent. We discuss the kinetic capacity of the ABC-type glucuronide transporter to explain net vacuolar flavone glucuronide accumulation in planta during rye primary leaf development and the possibility of an interaction of potential substrates at both the substrate binding and allosteric sites of the MRP transporter regulating the activity towards a certain substrate.