All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn 2؉ and Cd 2؉ , whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.Zinc is an essential element for all living organisms (1). Zinc chemistry is widely exploited to drive enzymatic catalysis, organize protein structures, and mediate macromolecular interactions (2). In known proteomes, zinc-containing metalloproteins account for ϳ10% of the total proteins (3). Zinc metabolism is also very high. In human, ϳ1% of the total body zinc content is replenished daily by the diet (4). The abundant zinc utilization and its rapid turnover necessitate highly efficient zinc uptake mechanisms by which cells accumulate zinc to a total concentration in the submillimolar range (5). The vast majority of cellular zinc is in complex with specific protein partners (6). Free zinc ions, on the other hand, must be strictly limited in the cytoplasm to prevent cytotoxic side effects (7). Zinc efflux transporters and intracellular buffering systems are evolved to maintain an extremely low level of cytoplasmic free zinc, probably in a femtomolar to picomolar range (8, 9). When a zinc supply is available in the extracellular medium, the free zinc concentrations in the cytoplasm are expected to be many orders of magnitude lower than the extracellular zinc concentrations (10, 11). The inward zinc concentration gradients would provide a powerful chemical driving force to draw extracellular zinc ions into the cytoplasm if a transmembrane zinc conduit would connect the external zinc availability and the high intracellular zinc binding capacity. Such a zinc-specific uptake channel has not heretofore been identified.A common assumption is that cellular zinc uptake is an active process mediated by metal transporters. In mammals, Zrt-, Irt-like protein (ZIP) 2 is the only zinc-specific uptake protein identified thus far (12). Although ZIPs supply zinc to meet cellular needs for growth, aberrant ZIP expressions have been linked to uncontrolled cell growth such as that occurring in cancer (13). Functionally, mammalian ZIPs promote zinc influx into the cytoplasm either from the extracellular medium or from zin...