Copper ion homeostasis in yeast is maintained through regulated expression of genes involved in copper ion uptake, Cu(I) sequestration, and defense against reactive oxygen intermediates. Positive and negative copper ion regulation is observed, and both effects are mediated by Cu(I)-sensing transcription factors. The mechanism of Cu(I) regulation is distinct for transcriptional activation versus transcriptional repression. Cu(I) activation of gene expression in S. cerevisiae and C. glabrata occurs through Cu-regulated DNA binding. The activation process involves Cu(I) cluster formation within the regulatory domain in Ace1 and Amt1. Cu(I) binding stabilizes a specific conformation capable of high-affinity interaction with specific DNA promoter sequences. Cu(I)-activated transcription factors are modular proteins in which the DNA-binding domain is distinct from the domain that mediates transcriptional activation. The all-or-nothing formation of the polycopper cluster permits a graded response of the cell to environmental copper. Cu(I) triggering may involve a metal exchange reaction converting Ace1 from a Zn(II)-specific conformer to a clustered Cu(I) conformer. The Cu(I) regulatory domain occurs in transcription factors from S. cerevisiae and C. glabrata. Sequence homologs are also known in Y. lipolytica and S. pombe, although no functional information is available for these candidate regulatory molecules. The presence of the Cu(I) regulatory domain in four distinct yeast strains suggests that this Cu-responsive domain may occur in other eukaryotes. Cu-mediated repression of gene expression in S. cerevisiae occurs through Cu(I) regulation of Mac1. Cu(I) binding to Mac1 appears to inhibit the transactivation domain. The Cu(I) specificity of this repression is likely to arise from formation of a polycopper thiolate cluster.