Alzheimer's disease is a neurodegenerative disorder in which the formation of amyloid-beta (A beta) aggregates plays a causative role. There is ample evidence that Cu(II) can bind to A beta and modulate its aggregation. Moreover, Cu(II) bound to A beta might be involved in the production of reactive oxygen species, a process supposed to be involved in the Alzheimer's disease. The native A beta 40 contains a high affinity binding site for Cu (II), which is comprised in the N-terminal portion. Thus, A beta 16 (amino acid 1-16 of A beta) has often been used as a model for Cu(II)-binding to monomeric A beta. The Cu(II)-binding to A beta is pH dependent and at pH 7.4, two different type of Cu(II) coordinations exist in equilibrium. These two forms are predominant at pH 6.5 and pH 9.0. In either form, a variety of studies show that the N-terminal Asp and the three His play a key role in the coordination, although the exact binding of these amino acids has not been addressed. Therefore, we studied the coordination modes of Cu(II) at pH 6.5 and 9.0 with the help of Fourier transform infrared (FTIR) spectroscopy. Combined with isotopic labeling of the amino acids involved in the coordination sphere, the data points toward the coordination of Cu(II) via the carboxylate of Asp1 at both pH values in a pseudobridging monovalent fashion. At low pH, His6 binds copper via N tau, while His13 and His14 are bound via N pi. At high pH, direct evidence is given on the coordination of Cu(II) via the N tau atom of His6. Additionally, this study clearly shows the effect of Cu(II) binding on the protonation state of the His residues where a proton displacement takes places on the nitrogen atoms of the imidazole ring.