Aggregation of the normal cellular prion protein, PrP, is important in the pathogenesis of prion disease. PrP binds glycosaminoglycan (GAG) and divalent cations, such as Cu(2+) and Zn(2+). Here, we report our findings that GAG and Cu(2+) promote the aggregation of recombinant human PrP (rPrP). The normal cellular prion protein has five octapeptide repeats. In the presence of either GAG or Cu(2+), mutant rPrPs with eight or ten octapeptide repeats are more aggregation prone, exhibit faster kinetics and form larger aggregates than wild-type PrP. When the GAG-binding motif, KKRPK, is deleted the effect of GAG but not that of Cu(2+) is abolished. By contrast, when the Cu(2+)-binding motif, the octapeptide-repeat region, is deleted, neither GAG nor Cu(2+) is able to promote aggregation. Therefore, the octapeptide-repeat region is critical in the aggregation of rPrP, irrespective of the promoting ligand. Furthermore, aggregation of rPrP in the presence of GAG is blocked with anti-PrP mAbs, whereas none of the tested anti-PrP mAbs block Cu(2+)-promoted aggregation. However, a mAb that is specific for an epitope at the N-terminus enhances aggregation in the presence of either GAG or Cu(2+). Therefore, although binding of either GAG or Cu(2+) promotes the aggregation of rPrP, their aggregation processes are different, suggesting multiple pathways of rPrP aggregation.