As an approach to develop both oligodendrocytic and astrocytic cell lines from adult human spinal cord, a cellular preparation of highly enriched oligodendrocytes and their precursors was infected with a replication-deficient retrovirus containing DNA sequences encoding the temperature-sensitive mutant of SV40 large T antigen. Six immortal cell lines were obtained. At both permissive (33 degrees C) and non-permissive (38.5 degrees C) temperatures, all cell lines were positive for vimentin, two demonstrated glial fibrillary acidic protein (GFAP) immunoreactivity, and none expressed oligodendrocyte or microglial markers. The 2 GFAP-positive cell lines [human spinal cord (HSC)2 and HSC6] were further characterized. Karyotype analysis revealed that both HSC2 and HSC6 cells showed gain of chromosomal material and structural chromosomal abnormalities. However, at non-permissive temperature both cell lines were indistinguishable from primary human astrocytes by a number of criteria. These properties included glutamine synthetase activity, Na(+)-dependent glutamate uptake, K+ flux, purine-evoked Ca2+ mobilization and entry, and the ability to support neurite outgrowth from embryonic rat retinal explants. The HSC2 and HSC6 cell lines may prove to be valuable models for studying the physiological properties of adult human astrocytes.