Bile acids were shown to be transported into barley mesophyll vacuoles. Uptake of the cholate conjugates taurocholate and glycocholate is strictly ATP-dependent. Uptake of taurocholate is a saturable process (Km = 40 microM) and is inhibited by vanadate but not by bafilomycin, a specific inhibitor of the vacuolar H(+)-ATPase. Together with the observation that the non-hydrolyzable ATP analog AMPPNP (5'-adenylyl beta,gamma-imidodiphosphate) does not stimulate, but rather inhibits, the ATP-dependent uptake of taurocholate, and that a 3-fold accumulation of the bile acid is observed in the presence of bafilomycin, these results suggest that taurocholate is transported into the vacuole by a primary active process as is the case for its canalicular secretion in rat liver (Nishida, T., Gatmaitan, Z., Che, M., and Arias, I. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6590-6594). Taurocholate uptake is inhibited by other bile acids and is slightly stimulated by glutathione S-conjugates. The different responses of the glutathione S-conjugate (Martinoia, E., Grill, E., Tommasini, R., Kreuz, K., and Amrhein, N. (1993) Nature 364, 247-249) and the taurocholate transporters, respectively, to substrates, oligomycin, GTP, and UTP suggest the presence of at least two ATPases specifically involved in the transport of conjugates across the tonoplast. As cholate and its conjugates have so far not been reported to occur in plants, the physiological function of the novel transport ATPase described here is presently unknown.