Understanding the metal-binding properties and solution states of metallo-chaperones is a key step in understanding how they function in metal ion transfer. Using spectroscopic, bioanalytical and biochemical methods, we have investigated the copper-binding properties and association states of the putative copper chaperone of Bacillus subtilis, CopZ, and a variant of the protein lacking the two cysteine residues of the MXCXXC copper-binding motif. We show that copper-free CopZ exists as a monomer, but that addition of copper(I) causes the protein to associate into homodimers. The nature of the copper(I)-CopZ complex is dependent on the level of copper loading, and we report the detection of three distinct forms, containing 0.5, 1.0 and 1.5 copper(I) ions per protein. The presence of excess dithiothreitol has a significant effect on copper(I) binding to CopZ, such that, in its presence, copper(I)-CopZ occurs mainly as a monomer species. Data for copper binding to the double-cysteine variant of CopZ are consistent with an essential role for these residues in tight copper binding in the wild-type protein. We conclude that the complex nature of copper(I) binding to CopZ may underpin mechanisms of protein-to-protein copper(I) transfer.