GEF1 is a gene in Saccharomyces cerevisiae, which encodes a putative voltage-regulated chloride channel. gef1 mutants have a defect in the high-affinity iron transport system, which relies on the cell surface multicopper oxidase Fet3p. The defect is due to an inability to transfer Cu⁺ to apoFet3p within the secretory apparatus. We demonstrate that the insertion of Cu into apoFet3p is dependent on the presence of Cl⁻. Cu-loading of apoFet3p is favored at acidic pH, but in the absence of Cl⁻ there is very little Cu-loading at any pH. Cl⁻ has a positive allosteric effect on Cu-loading of apoFet3p. Kinetic studies suggest that Cl⁻ may also bind to Fet3p and that Cu⁺ has an allosteric effect on the binding of Cl⁻ to the enzyme. Thus, Cl⁻ may be required for the metal loading of proteins within the secretory apparatus. These results may have implications in mammalian physiology, as mutations in human intracellular chloride channels result in disease.