The membrane-bound CzcA protein, a member of the resistance-nodulation-cell division (RND) permease superfamily, is part of the CzcCB2A complex that mediates heavy metal resistance in Ralstonia sp. CH34 by an active cation efflux mechanism driven by cation/proton antiport. The czcA gene was cloned into the plasmid pASK-IBA3 and the CzcA-strep-tag- fusion protein was purified to homogeneity after expression in Escherichia coli, reconstituted into detergent destabilized liposomes., and the kinetics of heavy metal transport by CzcA was determined. CzcA is composed of 12 transmembrane α-helices and two large periplasmic domains. Two conserved aspartate and a glutamate residue in one of these transmembrane spans are essential for heavy metal resistance and proton/cation antiport but not for facilitated diffusion of cations. The velocity of metal cation uptake was determined for all three metals. For zinc and cobalt, the substrate saturation curve was sigmoidal. For zinc a Vmax value of 385 s-1 was calculated from the velocites at 10 and 5 mM zinc, and this velocity was used for a Hill plot yielding a cooperativity constant n=2 and a K50 of 6.6 mM. For cobalt, using the velocity measured at 50 mM, a Hill plot was performed yielding a cooperativity constant n=2 and a K50 of 18.5 mM. Cadmium transport by CzcA was even slower than cobalt uptake. No uptake could be detected at concentrations lower than 1 mM or higher than 5 mM. In this narrow range of substrate concentration a Lineweaver-Burk plot was linear and yielded a Vmax of 28 s-1 and a Km of 7.7 mM. The mutated proteins (CzcA-D402N and CzcA-D408N) also were reconstituted into detergent destabilized liposomes, and the kinetics of heavy metal transport by the mutated CzcA proteins were determined. When they were compared with the wild type protein, all three proteins displayed the rapid facilitated diffusion of zinc, but the mutant proteins were no longer able to catalyze the slower proton/zinc antiport. Generalization of the resulting model for the function of CzcA as a two-channel pump might help to explain the function of RND proteins.