ABSTRACT Copper homeostasis within the cell is established and preserved by different mechanisms. Changes in gene expression constitute a way of maintaining this homeostasis. In Schizosaccharomyces pombe , the Cuf1 transcription factor is critical for the activation of copper transport gene expression under conditions of copper starvation. However, in the presence of elevated intracellular levels of copper, the mechanism of Cuf1 inactivation to turn off gene expression remains unclear. In this study, we provide evidence that inactivation of copper transport gene expression by Cuf1 is achieved through a copper-dependent, cytosolic retention of Cuf1. We identify a minimal nuclear localization sequence (NLS) between amino acids 11 to 53 within the Cuf1 N terminus. Deletion of this region and specific mutation of the Lys ¹³ , Arg ¹⁶ , Arg ¹⁹ , Lys ²⁴ , Arg ²⁸ , Lys ⁴⁵ , Arg ⁴⁷ , Arg ⁵⁰ , and Arg ⁵³ residues to alanine within this putative NLS is sufficient to abrogate nuclear targeting of Cuf1. Under conditions of copper starvation, Cuf1 resides in the nucleus. However, in the presence of excess copper as well as silver ions, Cuf1 is sequestered in the cytoplasm, a process which requires the putative copper binding motif, ³²⁸ Cys-X-Cys-X ₃ -Cys-X-Cys-X ₂ -Cys-X ₂ -His ³⁴² (designated C-rich), within the C-terminal region of Cuf1. Deletion of this region and mutation of the Cys residues within the C-rich motif result in constitutive nuclear localization of Cuf1. By coexpressing the Cuf1 N terminus with its C terminus in trans and by using a two-hybrid assay, we show that these domains physically interact with each other in a copper-dependent manner. We propose a model wherein copper induces conformational changes in Cuf1 that promote a physical interaction between the Cuf1 N terminus and the C-rich motif in the C terminus that masks the NLS. Cuf1 is thereby sequestered in the cytosol under conditions of copper excess, thereby extinguishing copper transport gene expression.