Complex formation processes between the 39-mer residue peptide fragment of human prion protein, HuPrP(76-114), and copper(II) ions have been studied by potentiometric, UV-vis, circular dichroism (CD), electron paramagnetic resonance, and electrospray ionization mass spectrometry methods. This peptide consists of 39 amino acid residues and contains two histidines (His77 and His85) belonging to the octarepeat domain and two histidines (His96 and His111) outside this domain. It was found that HuPrP(76-114) is able to bind 4 equiv of metal ions and all histidyl residues are independent, except nonequivalent metal binding sites in the oligonuclear species. Imidazole nitrogen donor atoms are the primary and exclusive metal binding sites below pH 5.5 in the form of various macrochelates. The macrochelation slightly suppresses, but cannot prevent, the deprotonation and metal ion coordination of amide functions, resulting in the formation of (N-im,N-), (N-im, N-,N-), and (N-im, N-, N-, N-)-coordinated copper(II) complexes in the pH range from 5.5 to 9. CD spectroscopy results gave clear evidence for the differences in the metal binding affinity of the histidyl sites according to the following order: His111 > His96 >> His77 similar to His85. Among the oligonuclear complexes, the formation of di- and tetranuclear species seems to be favored over the trinuclear ones, at pH values beyond the physiological ones. This phenomenon was not observed in the complex formation reactions of HuPrP(84-114), a peptide fragment containing only one histidyl residue from the octarepeat. As a consequence, the data support the existence of cooperativity in the metal binding ability of this peptide probably due to the presence of two octarepeat sequences of the dimeric octarepeat domain of HuPrP(76-114) at basic pH values.