Amyloid-beta (Abeta) peptide is the principal constituent of plaques associated with Alzheimer's disease and is thought to be responsible for the neurotoxicity associated with the disease. Metal ions have been hypothesized to play a role in the formation and neurotoxicity of aggregates associated with Alzheimer's disease (Bush, A. I.; et al. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 11934). Elucidation of the chemistry through which transition-metal ions participate in the assembly and toxicity of Abeta oligomers is important to drug design efforts if inhibition of Abeta containing bound metal ions becomes a treatment for Alzheimer's disease. In this paper, we report electron paramagnetic resonance (EPR) spectroscopic characterization of Cu2+ bound to soluble and fibrillar Abeta. Addition of stoichiometric amounts of Cu2+ to soluble Abeta produces an EPR signal at 10 K with observable Cu2+ hyperfine lines. A nearly identical spectrum is observed for Abeta fibrils assembled in the presence of Cu2+. The EPR parameters are consistent with a Type 2 Cu2+ center with three nitrogen donor atoms and one oxygen donor atom in the coordination sphere of Cu2+: g, = 2.26 and A = 174 +/- 4 G for soluble Abeta with Cu2+, and g(II), = 2.26 and A = 175 +/- 1 G for Abeta fibrils assembled with Cu2+. Investigation of the temperature dependence of the EPR signal for Cu2+ bound to soluble Abeta or Cu2+ in fibrillar Abeta shows that the Cu2+ center displays normal Curie behavior, indicating that the site is a mononuclear Cu2+ site. Fibrils assembled in the presence of Cu2+ contain one Cu2+ ion per peptide. These results show that the ligand donor atom set to Cu2+ does not change during organization of A/3 monomers into fibrils and that neither soluble nor fibrillar forms of Abeta(1-40) with Cu2+ contain antiferromagnetically exchange-coupled binuclear Cu2+ sites in which two Cu2+ ions are bridged by an intervening ligand.