Prion diseases are characterized by the conversion of the cellular prion protein (PrPC) to a disease-specific aggregated isoform (PrPSc). We have shown that Mn2+ ions amplify aggregation, whereas Cu2+ has an inhibitory effect. To characterize Mn2+-induced aggregates, we used cross-correlation analysis as well as scanning for intensely fluorescent targets in an SDS-dependent aggregation assay with fluorescently labeled PrP. We found that the effect of Mn2+ was mainly due to the association of preformed PrP oligomers to larger aggregates, rapidly reversible by EDTA, and independent of the histidine-dependent copper-binding sites of PrP, suggesting that Mn2+ induces reversible intermolecular binding. In contrast, the inhibitory effect of Cu2+ required binding to histidine-containing binding sites, indicating that binding of copper affects the structure of PrPC which in turn modifies the susceptibility to manganese and the ability to aggregate. These findings suggest that copper and manganese may also affect prion propagation in vivo. (c) 2005 Elsevier Inc. All rights reserved.