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The cation diffusion facilitator (CDF) family represents a class of ubiquitous metal transporters. Inactivation of a CDF in Schizosaccharomyces pombe, Zhf, causes drastically different effects on the tolerance toward various metals. A deletion mutant is Zn2+/Co2+-hypersensitive yet displays significantly enhanced Cd2+ and Ni2+ tolerance. Accumulation of zinc, cobalt, and cadmium is reduced in mutant cells. Non-vacuolar zinc content, as measured by analytical electron microscopy, is lower in zhf− cells compared with wild-type cells in the presence of elevated Zn2+concentrations. The protective effect against cadmium toxicity is independent of the phytochelatin detoxification pathway. Phytochelatin synthase-deficient cells show extremely enhanced (about 200-fold) cadmium tolerance when zhf is disrupted. Immunogold labeling indicates endoplasmic reticulum (ER) localization of Zhf. Electron spectroscopic imaging shows that accumulation of zinc coincides with Zhf localization, demonstrating a major role of the ER for metal storage and the involvement of Zhf in cellular zinc homeostasis. Also, these observations indicate that Cd2+ions exert their toxic effects on cellular metabolism in the ER rather than in the cytosol.