Amyloid β (Aβ) is a Cu-binding peptide that plays a key role in the pathology of Alzheimer's disease. A recent report demonstrated that Aβ disrupts the Cu-dependent interaction between cellular prion protein (PrP C ) and N-methyl-D-aspartate receptor (NMDAR), inducing overactivation of NMDAR and neurotoxicity. In this context, it has been proposed that Aβ competes for Cu with PrP C ; however, there is no spectroscopic evidence to support this hypothesis. Prion protein (PrP) can bind up to six Cu(II) ions: from one to four at the octarepeat (OR) region, producing low-and high-occupancy modes, and two at the His96 and His111 sites. Additionally, PrP C is cleaved by αsecretases at Lys110/His111, yielding a new Cu(II)-binding site at the α-cleaved His111. In this study, the competition for Cu(II) between Aβ(1−16) and peptide models for each Cu-binding site of PrP was evaluated using circular dichroism and electron paramagnetic resonance. Our results show that the impact of Aβ(1−16) on Cu(II) coordination to PrP is highly site-specific: Aβ(1−16) cannot effectively compete with the low-occupancy mode at the OR region, whereas it partially removes the metal ion from the high-occupancy modes and forms a ternary OR-Cu(II)-Aβ(1−16) complex. In contrast, Aβ(1−16) removes all Cu(II) ions from the His96 and His111 sites without formation of ternary species. Finally, at the α-cleaved His111 site, Aβ(1−16) yields at least two different ternary complexes depending on the ratio of PrP/ Cu(II)/Aβ. Altogether, our spectroscopic results indicate that only the low-occupancy mode at the OR region resists the effect of Aβ, while Cu(II) coordination to the high-occupancy modes and all other tested sites of PrP is perturbed, by either removal of the metal ion or formation of ternary complexes. These results provide important insights into the intricate effect of Aβ on Cu(II) binding to PrP and the potential neurotoxic mechanisms through which Aβ might affect Cu-dependent functions of PrP C , such as NMDAR modulation.