Aggregation of alpha-synuclein (alpha-syn), a process that generates oligomeric intermediates, is a common pathological feature of several neurodegenerative disorders. Despite the potential importance of the oligomeric alpha-syn intermediates in neuron function, their biochemical properties and pathobiological functions in vivo remain vastly unknown. Here we used two-dimensional analytical separation and an array of biochemical and cell-based assays to characterize alpha-syn oligomers that are present in the nervous system of A53T alpha-syn transgenic mice. The most prominent species identified were 53 A detergent-soluble oligomers, which preceded neurological symptom onset, and were found at equivalent amounts in regions containing alpha-syn inclusions as well as histologically unaffected regions. These oligomers were resistant to SDS, heat, and urea but were sensitive to proteinase-K digestion. Although the oligomers shared similar basic biochemical properties, those obtained from inclusion-bearing regions were prominently reactive to antibodies that recognize oxidized alpha-syn oligomers, significantly accelerated aggregation of alpha-syn in vitro, and caused primary cortical neuron degeneration. In contrast, oligomers obtained from non-inclusion-bearing regions were not toxic and delayed the in vitro formation of alpha-syn fibrils. These data indicate that specific conformations of alpha-syn oligomers are present in distinct brain regions of A53T alpha-syn transgenic mice. The contribution of these oligomers to the development of neuron dysfunction appears to be independent of their absolute quantities and basic biochemical properties but is dictated by the composition and conformation of the intermediates as well as unrecognized brain-region-specific intrinsic factors.