Previous studies have shown that alpha-synuclein (alpha-Syn) aggregates derived from the brains of patients with Parkinson's disease (PD) and multiple system atrophy (MSA) exhibit different phosphorylation, cytotoxicity, and seeding activity. However, the mechanism underlying the differences remains poorly understood. Here, recombinant human alpha-Syn was incubated in the plasma of patients with PD and MSA, and the oligomers formed in the plasma (PD-O-alpha-Syn and MSA-O-alpha-Syn) were purified and analyzed for their phosphorylation, cytotoxicity and seeding activity. In vitro assays revealed that both PD-O-alpha-Syn and MSA-O-alpha-Syn were phosphorylated at serine 129. However, the phosphorylation degree of MSA-O-alpha-Syn was significantly higher than that of PD-O alpha-Syn. In addition, MSA-O-alpha-Syn exhibited stronger cytotoxicity and seeding activity compared with PD-O-alpha-Syn. In vivo experiments showed that mice receiving intrastriatal inoculation of MSA-O-alpha-Syn developed more severe motor dysfunction and dopaminergic degeneration than mice receiving intrastriatal inoculation of PD-O-alpha-Syn. Compared with the mice inoculated with PD-O-alpha-Syn, the mice inoculated with MSA-O-alpha-Syn accumulated more phosphorylated and oligomerized alpha-Syn in the striatum and brain regions (substantia nigra, hippocampus and prefrontal cortex) away from the inoculated site. The results obtained suggest that alpha-Syn oligomers formed in PD and MSA plasma are different in phosphorylation, cytotoxicity, and seeding activity.