Glial fibrillary acidic protein (GFAP) was present in cell cultures derived from human fetal brain tissue as determined by indirect immunofluorescence (IF) and immunoperoxidase (IP) staining using rabbit anti-human GFAP antisera. The IF and IP techniques were comparable in localizing the cytoplasmic distribution and the frequent perinuclear concentration of GFAP in brain cells. The horseradish peroxidase technique was more sensitive and a 1:20–1:40 dilution of anti-GFAP serum could be applied in the initial step of the peroxidase staining as compared to a 1:10 dilution of anti-GFAP serum for IF staining. Sequential studies of subcultivated human fetal brain cell lines by these techniques indicated that some brain cell lines become GFAP-negative rapidly, whereas other cell lines remain GFAP-posifive for no less than ten subcultivations in vitro. GFA protein was never present in any PMLSV40-transformed human brain cells.