Several cultures established from biopsies of apparently normal adult human glial material showed no cells positive for glial fibrillary acidic protein (GFA) when examined after seven or more cumulative population doublings (CPD), although the established glioma line U251 MG showed approximately 3% GFA-positive cells, and U333 CG/343 MG clone 3 showed greater than 98% GFA-positive cells. Both the human glia delivered cultures and the glioma lines were positive when assayed with sera specific for vimentin. We therefore investigated the expression of GFA as a function of cumulative population doublings after the establishment of primary cultures. Under our experimental conditions, although GFA-positive cells were clearly present in the primary cultures accounting for some 3%-14% of the cells present, the GFA marker was subsequently lost, and the proliferating cultures expressed only the vimentin type of intermediate filament. Those cells that were GFA-positive also appeared to be vimentin-positive. GFA expression was not reinduced in cultures that had lost the GFA marker by treatment with dibutyryl cyclic AMP. We discuss two alternate hypotheses for the origin of the GFA-negative cells: (1) the cultures area of astrocyte origin but lost the ability to express GFA on culturing; (2) the cultures originate from cells of nonastrocyte origin present in the primary material.