αB-crystallin, a major protein of the vertebrate lens and a member of the small heat shock protein family, is expressed in non-lenticular tissues, including the central nervous system, where it is found mainly in glia. In Rosenthal fibers (RF), astrocytic inclusions that accumulate in Alexander's Disease, αB-crystallin is found with hsp27 and skeins of intermediate filaments (IF) of the GFAP and vimentin types. We have investigated the association between IF and αB-crystallin in a human astrocytoma cell line, U-373MG, which expresses αB-crystallin. Cytoskeletal preparations contained αB-crystallin, and a filamentous pattern in which αB-crystallin co-localized with GFAP and vimentin by double label immunofluorescence. Immuno-electronmicroscopy confirmed the localization to IF. GFAP isolated from bovine brain and re-assembled, was associated with αB-crystallin. Thus, a proportion of αB-crystallin in astroglia is associated with IF, and this association may be critical in the formation of RF.