A series of tripodal ligands L derived from nitrilotriacetic acid (NTA) and extended by three converging metal-binding cysteine chains were previously found to bind selectively copper(I) both in vitro and in vivo. The ligands L' (ester) and If (amide) were demonstrated to form copper(I) species with very high affinities, close to that reported for the metal-sequestering metallothioneins (MTs; log KCu-MT approximate to 19). Here, an in-depth study by Cu K-edge X-ray absorption -spectroscopy (XAS) was performed to completely characterize the copper(I) coordination sphere in the complexes, previously evidenced by other physicochemical analyses. The X-ray absorption near-edge structure ()CANES) spectra shed light on the equilibrium between a mononuclear complex and. a cluster for both L' (ester) and L2 (amide). The exclusive symmetric CuS3 geometry adopted in the mononuclear complexes (Cu S 2.23 A) was clearly demonstrated by extended X-ray absorption fine structure (EXAFS) analyses. The EXAFS analyses also proved that the clusters are organized on a symmetric CuS3 core (Cu S RI: 2.26 A) and interact with three nearby copper atoms (Cu Cu P.: 2.7 A), consistent with the Cu6S9-type clusters previously characterized by pulsed gradient spin echo NMR spectroscopy. XAS data obtained for other architectures based on the NTA template (L3 acid, L4 without a functionalized carbonyl group, etc) demonstrated the formation of polymetallic species only, which evidence the necessity of the proximal ester or amide group to stabilize the CuS3 mononuclear species. Finally, XAS was demonstrated to be a powerful method to quantify the equilibrium between the two copper(I) environments evidenced with LI and 1.,2 at different copper concentrations and to determine the equilibrium constants between these two complexes.