It is well established that the prion protein (PrP) contains metal ion binding sites with specificity for copper. Changes in copper levels have been suggested to influence incubation time in experimental prion disease. Therefore, we studied the effect of heavy metal ions (Cu2+, Mn2+, Ni2+, Co2+, and Zn2+) in vitro in a model system that utilizes changes in the concentration of SDS to induce structural conversion and aggregation of recombinant PrP. To quantify and characterize PrP aggregates, we used fluoreseently labelled PrP and cross-correlation analysis as well as scanning for intensely fluorescent targets in a confocal single molecule detection system. We found a specific strong pro-aggregatory effect of Mn2+ at low micromolar concentrations that could be blocked by nanomolar concentration of Cu2+. These findings suggest that metal ions such as copper and manganese may also affect PrP conversion in vivo. (C) 2004 Elsevier Inc. All rights reserved.