The CLC family of chloride channels and transporters is a functionally diverse group of proteins important in a wide range of physiological processes. ClC-4 and ClC-5 are localized to endosomes and seem to play roles in the acidification of these compartments. These proteins were recently shown to function as Cl(-)/H(+) antiporters. However, relatively little is known about the detailed mechanism of CLC-mediated Cl(-)/H(+) antiport, especially for mammalian isoforms. We attempted to identify molecular tools that might be useful in probing structure-function relationships in these proteins. Here, we record currents from human ClC-4 (hClC-4) expressed in Xenopus oocytes, and find that Zn(2+) inhibits these currents, with an apparent affinity of approximately 50 microM. Although Cd(2+) has a similar effect, Co(2+) and Mn(2+) do not inhibit hClC-4 currents. In contrast, the effect of Zn(2+) on the ClC-0 channel, Zn(2+)-mediated inhibition of hClC-4 is minimally voltage-dependent, suggesting an extracellular binding site for the ion. Nine candidate external residues were tested; only mutations of three consecutive histidine residues, located in a single extracellular loop, significantly reduced the effect of Zn(2+), with one of these making a larger contribution than the other two. An analogous tri-His sequence is absent from ClC-0, suggesting a fundamentally different inhibitory mechanism for the ion on hClC-4. Manipulations that alter transport properties of hClC-4, varying permeant ions as well as mutating the "gating glutamate", dramatically affect Zn(2+) inhibition, suggesting the involvement of a heretofore unexplored part of the protein in the transport process.