In Alzheimer’s disease, copper binds to amyloid beta (Aβ) peptide and generates oxidative stress. The coordination of histidine (His) residues to Cu2+ is still uncertain. We studied Cu2+ binding to Aβ1−16 peptide using the diethyl pyrocarbonate (DEPC) assay and mass spectrometry. Our results show that only one His is involved in Cu2+ coordination, which is identified as His6 using mass spectral studies. Novel nickel displacement studies have further supported the proposal that the Cu2+ binding site of Aβ1−16 peptide resembles the ATCUN motif of human serum albumin.