Zinc is an essential cofactor required for the function of approximately 8% of the yeast and 10% of the human proteome. All of the "small Tim" proteins of the mitochondrial intermembrane space contain a strictly conserved "twin CX(3)C" zinc finger motif, which can bind zinc ions in the Cys-reduced form. We have shown previously that although disulfide bond formation is essential for the function of these proteins in mitochondria, only reduced proteins can be imported into mitochondria (Lu, H., Allen, S., Wardleworth, L., Savory, P., and Tokatlidis, K. (2004) J. Biol. Chem. 279, 18952-18958 and Morgan, B., and Lu, H. (2008) Biochem. J. 411, 115-122). However, the role of zinc during the import of these proteins is unclear. This study shows that the function of zinc is complex. It can play a thiol stabilizer role preventing oxidative folding of the small Tim proteins and maintaining the proteins in an import-competent form. On the other hand, zinc-bound forms cannot be imported into mitochondria efficiently. Furthermore, our results show that zinc is a powerful inhibitor of Erv1, an essential component of the import pathway used by the small Tim proteins. We propose that zinc plays a chaperone-like role in the cytosol during biogenesis of the small Tim proteins and that the proteins are imported into mitochondria through the apo-forms.