In Lewy body diseases—including Parkinson's disease, without or with dementia, dementia with Lewy bodies, and Alzheimer's disease with Lewy body co-pathology1—α-synuclein (α-Syn) aggregates in neurons as Lewy bodies and Lewy neurites2. By contrast, in multiple system atrophy α-Syn accumulates mainly in oligodendrocytes as glial cytoplasmic inclusions (GCIs)3. Here we report that pathological α-Syn in GCIs and Lewy bodies (GCI-α-Syn and LB-α-Syn, respectively) is conformationally and biologically distinct. GCI-α-Syn forms structures that are more compact and it is about 1,000-fold more potent than LB-α-Syn in seeding α-Syn aggregation, consistent with the highly aggressive nature of multiple system atrophy. GCI-α-Syn and LB-α-Syn show no cell-type preference in seeding α-Syn pathology, which raises the question of why they demonstrate different cell-type distributions in Lewy body disease versus multiple system atrophy. We found that oligodendrocytes but not neurons transform misfolded α-Syn into a GCI-like strain, highlighting the fact that distinct α-Syn strains are generated by different intracellular milieus. Moreover, GCI-α-Syn maintains its high seeding activity when propagated in neurons. Thus, α-Syn strains are determined by both misfolded seeds and intracellular environments.