The malate carrier of barley (Hordeum vulgare L.) mesophyll vacuoles was highly purified by chromatography on hydroxyapatite followed by affinity-chromatography using 5-amino-1,2,3-benzenetricarboxylic acid as ligand. The carrier, reconstituted in asolectin liposomes, had properties similar to those described previously for the carrier in intact vacuoles (Martinoia, E., Flügge, U.I., Kaiser, G., Heber, U. and Heldt, H.W. (1985) Biochim. Biophys. Acta 806, 311-319). The apparent Km for malate uptake was 2-3 mM, and the uptake was inhibited by other carboxylic acids (preferentially tricarboxylic). The sulfhydryl reagent, p-chloromercuribenzenesulfonate, as well as the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, also inhibited malate uptake. The transport was dependent on the membrane potential with an optimum at about 35 mV.