Aggregation and cytotoxicity of misfolded α-synuclein is postulated to be crucial in the disease process of neurodegenerative disorders such as Parkinson's disease and DLB (dementia with Lewy bodies). In this study, we detected misfolded and aggregated α-synuclein in a Triton X-100 insoluble fraction as well as a high molecular weight product by gel electrophoresis of temporal neocortex from DLB patients but not from controls. We also found similar Triton X-100 insoluble forms of α-synuclein in an α-synuclein transgenic mouse model and in an in vitro model of α-synuclein aggregation. Introducing the molecular chaperone Hsp70 into the in vivo model by breeding α-synuclein transgenic mice with Hsp70-overexpressing mice led to a significant reduction in both the high molecular weight and detergent-insoluble α-synuclein species. Concomitantly, we found that Hsp70 overexpression in vitro similarly reduced detergent-insoluble α-synuclein species and protected cells from α-synuclein-induced cellular toxicity. Taken together, these data demonstrate that the molecular chaperone Hsp70 can reduce the amount of misfolded, aggregated α-synuclein species in vivo and in vitro and protect it from α-synuclein-dependent toxicity.