The HAHI metallochaperone is a key protein implicated in copper homeostasis in human cells. Using as solid-phase based assay completed with Biacore studies, we provided evidence that HAHI forms homo-dimers in the presence of copper. Biacore analysis allowed us to determine the kinetic parameters of this interaction, characterised by an apparent affinity constant of 6 muM. Moreover, we demonstrated that copper-loaded HAHI interacts independently with each of the six individual metal-binding domains of the copper- translocating Menkes ATPase. Finally, the homo-dimerisation of the metallochaperone was confirmed in living cells by using fluorescence resonance energy transfer. Results have been discussed in the context of intracellular copper control. (C) 2004 Elsevier Inc. All rights reserved.