The cytosolic C-terminal domain of the membrane copper transporter Ctr1 from the yeast Saccharomyces cerevisiae, Ctr1c, was expressed in E. coli as an oxygen-sensitive soluble protein with no significant secondary structure. Visible-UV spectroscopy demonstrated that Ctr1c bound four Cu(I) ions, structurally identified as a CuI4(μ-S-Cys)6 cluster by Xray absorption spectroscopy. This was the only metalated form detected by electrospray ionization mass spectrometry. An average dissociation constant KD = (K1K2K3K 4)1/4 = 10-19 for binding of Cu(I) to Ctr1c was estimated via competition with the ligand bathocuproine disulfonate bcs (β2 = 1019.8). Equivalent experiments for the yeast chaperone Atx1 and an N-terminal domain of the yeast Golgi pump Ccc2, which both bind a single Cu(I) ion, provided similar KD values. The estimates of KD were supported by independent estimates of the equilibrium constants Kex for exchange of Cu(I) between pairs of these three proteins. It is apparent that, in vitro, the three proteins buffer "free" Cu(I) concentrations in a narrow range around 10-19 M. The results provide quantitative support for the proposals that, in yeast, (a) "free" copper concentrations are very low in the cytosol and (b) the Cu(I) trafficking gradient is shallow along the putative Ctr1c → Atx1 → Ccc2n metabolic pathway. In addition, both Ctr1c and its copper-responsive transcription factor Mac1 contain similar clusters which may be important in signaling copper status in yeast.