Prion diseases are neurodegenerative disorders that result from conformational transformation of a normal cell surface glycoprotein, PrP(C), into a pathogenic isoform, PrP(Sc). Although the normal physiological function of PrP(C) has remained enigmatic, the recent observation that the protein binds copper ions with micromolar affinity suggests a possible role in brain copper metabolism. In this study, we have used mice that express 0, 1, and 10 times the normal level of PrP to assess the effect of PrP expression level on the amount of brain copper and on the properties of two brain cuproenzymes. Using mass spectrometry, we find that the amount of ionic copper in subcellular fractions from brain is similar in all three lines of mice. In addition, the enzymatic activities of Cu-Zn superoxide dismutase and cytochrome c oxidase in brain extracts are similar in these groups of animals, as is the incorporation of (64)Cu into Cu-Zn superoxide dismutase both in cultured cerebellar neurons and in vivo. Our results differ from those of another set of published studies, and they require a re-evaluation of the role of PrP(C) in copper metabolism.