The amyloid-beta (A beta) peptide is a cleavage product of the amyloid precursor protein and has been implicated as a central player in Alzheimer's disease. The N-terminal end of A beta is variable, and different proportions of these variable-length A beta peptides are present in healthy individuals and those with the disease. The N-terminally truncated form of A beta starting at position 4 (A beta(4-x)) has a His residue as the third amino acid (His6 using the formal numbering). The N-terminal sequence Xaa-Xaa-His is known as an amino terminal copper and nickel binding motif (ATCUN), which avidly binds Cu(II). This motif is not present in the commonly studied A beta(1-x) peptides. In addition to the ATCUN site, A beta(4-x) contains an additional metal binding site located at the tandem His residues (his-His at His13 and 14) which is also found in other isoforms of A beta. Using the ATCUN and bis-His motifs, the A beta(4-x) peptide is capable of binding multiple metal ions simultaneously. We confirm that Cu(II) bound to this particular ATCUN site is redox silent, but the second Cu(II) site is redox active and can be readily reduced with ascorbate. We have employed surrogate metal ions to block copper coordination at the ATCUN or the tandem His site in order to isolate spectral features of the copper coordination environment for structural characterization using extended X-ray absorption fine structure (EXAFS) spectroscopy. This approach reveals that each copper coordination environment is independent in the Cu(2)A beta(4-x) state. The identification of two functionally different copper binding environments within the A beta(4-x) sequence may have important implications for this peptide in vivo.