The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to "catalyze" the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Abeta) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Abeta. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Abeta was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Abeta in a concentration-dependent but ATP-independent manner. Inhibition of Abeta fibrillization by Hsp104 is observable up to Hsp104/Abeta stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Abeta amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Abeta protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Abeta. Together, these findings suggest that the strong inhibition of Abeta fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation.