A rapid procedure was developed for the purification and reconstitution into proteoliposomes of the tonoplast H&43 -translocating ATPase from the Crassulacean acid metabolism (CAM) plant Kalanchoédaigremontiana. It involved the fractionation of crude tonoplast membranes with the detergent Triton X-114, resolubization of the ATPase with octyl glucoside in the presence of an optimized lipid mixture and formation of liposomes on removal of detergent by gel filtration. The enzyme could be further purified by sedimentation through glycerol gradients. It contained polypeptides of apparent molecular mass 72, 57, 48, 42, 39, 33 and 16 kDa; the smallest of these was labelled by [¹⁴C]-dicyclohexylcarbodiimide. There was no evidence for the presence of any large subunits. In these proteolipsomes, ATP hydrolysis and H+ -translocation were measured independently, by a coupled enzyme assay and by quenching of the fluorescence of a permanent weak base, respectively. The kinetic properties of the reconstitued plant ATPase were studied in detail. Rate equations derived from theoretical models of the enzyme's behaviour were fitted to experimental data by weighted non-linear regression, using a computer program that calculated the kinetic parameters that accorded to the optimal fit. The dependence of the rate of H+ -translocation on the concentration of MgATP was well fitted by the Michaelis equation, with the K_m value about 30 μM. ATP could be replaced by dATP, ITP, GTP, UTP or CTP and Mg^2+ by Mn^2+ or Ca^2+ kinetic parameters for these substrates were determined. In contrast hydrolysis of MgATP showed complex kinetics which suggested either negative cooperativity between nucleotide-binding sites, or the presence of two non-interacting catalytic sites. Both the hydrolytic and the H^&43 -translocating activities of the proteoliposomes were inhibited by nitrate, though not in parallel, the latter activity being more sensitive. Both activities were inhibited in parallel by bafilomycin A_1, which did not produce complete inhibition; the bafilomycin-insensitive component had complex ATPase kinetics similar to those of the uninhibited enzyme. ADP behaved as an allosteric inhibitor of the ATPase, inducing apparent cooperativity in saturation with MgATP, together with a reduction in V_max.