The expression of glial fibrillary acidic protein (GFAP) in continuous glioma and non-glioma cell lines was evaluated by molecular hybridization and immunocytochemistry. RNA transcripts from the GFAP gene were detected in two of six cell lines by Northern blot analysis of cellular RNAs using a cloned mouse GFAP cDNA probe. The probe recognized a single GFAP RNA species of 2.7 kilobases under high-stringency washing conditions. The presence of GFAP transcripts as determined by molecular hybridization corresponded to the immunocytochemical detection of GFAP using two different monoclonal antibodies to this protein. These data indicate that GFAP expression in human cells can be detected by molecular hybridization using a murine GFAP cDNA probe, illustrating the high degree of inter-species conservation of GFAP. Molecular hybridization represents a reliable and sensitive method for the detection of GFAP expression in vitro, which can be used in conjunction with or as an alternative to immunocytochemistry.