Multiple system atrophy (MSA) is characterized by the formation of oligodendroglial cytoplasmic inclusions (GCIs) consisting of a-synuclein filaments. aB-crystallin, a small chaperone protein that binds to unfolded proteins and inhibits aggregation, has been documented in GCIs. We investigated the relative abundance and speciation of aB-crystallin in GCIs in MSA brains. We also examined the influence of aB-crystallin on the formation of cytoplasmic inclusions in cultured glial cells. Immunohistochemistry and confocal microscopy revealed aBcrystallin is a prominent component of GCIs, more abundant than in Lewy bodies in Lewy body dementia. One- and two-dimensional gel electrophoresis and mass spectrometric analysis of GCIs immunopurified from MSA brains indicated that aB-crystallin is a major protein component with multiple post-translationally modified species. In cultured C6 glioma cells treated with the proteasomal inhibitor, lactacystin, to induce accumulation of ubiquitinated proteins, a subset of cells showed increased cytoplasmic staining for aB-crystallin. Proteasomeinhibited cells transfected with GFP-tagged a-synuclein resulted in ubiquitin- and aB-crystallin-positive aggregates resembling GCIs in MSA brains. Our results indicate that aB-crystallin is a major chaperone in MSA, and suggest a role of the protein in the formation of inclusion bodies in glial cells.