The expression of glial fibrillary acidic protein (GFAP), vimentin and fibronectin (Fn) was studied in cells cultured from human glioma and fetal brain by indirect immunofluorescence (IIF) microscopy and multiple labelling experiments. In the primary cultures a major part (20%–70%) of the cells usually displayed both GFAP and vimentin and the rest of the cells only vimentin. A prominent variation in GFAP and vimentin fluorescence intensity sometimes made interpretation of double IIF stainings difficult. However, occasional GFAP-positive cells appeared vimentin negative in primary glioma cultures, whereas in fetal brain primary cultures cells that were preferentially GFAP positive also showed at least a weak vimentin immunoreactivity. Only a fraction of the cells, roughly corresponding to the GFAP-negative cells, were Fn positive in the primary cultures. As judged by double IIF, the GFAP-positive cells were usually Fn negative, while the Fn-positive cells were vimentin positive. This could also be demonstrated in triple IIF experiments. During serial subcultivation the amount of cells expressing GFAP decreased, while the number of Fn-positive cells increased. By the third to fourth passage GFAP positivity was usually lost, all cells expressed vimentin and most cells also Fn. The results of the present study demonstrate a general coexpression of GFAP and vimentin in cultured astroglial cells, in addition to cells expressing only vimentin. Interestingly, occasional glioma cells seem to contain GFAP as the only intermediate filament protein as detected by immunocytochemistry. Such cells, however, are seen only in primary cultures. Cells coexpressing GFAP and vimentin are also rapidly lost upon subculture, whereas immunocytochemical Fn positivity correspondingly increases.