Primary cultures of human fetal brain cells were transfected with plasmid DNA pMK16, containing an origin-defective mutant of simian virus 40 (SV40). Several weeks after DNA treatment, proliferation of glial cells was evident in the culture, allowing passage of the cells at low split ratios. Initially, only 10% of the cells demonstrated nuclear fluorescence staining using a hamster tumor antibody to the SV40 T protein. By the sixth passage, however, 100% of the cells reacted positively to the same antibody. During these early passages, the cells designated SVG began growing very rapidly and acquired a homogeneous morphology. Cell division required only low serum concentrations, was not contact-inhibited, and remained anchorage dependent. These characteristics of the SVG cells have been stable through 25 passages or approximately equal to 80 cell generations. The SV40 T protein is continuously produced in the cells and can direct the replication of DNA inserts in the pSV2 vector, determined by in situ hybridization using biotin-labeled DNA probes, which contains the SV40 replication origin. More importantly, SVG cells support the multiplication of the human papovavirus JCV at levels comparable to primary cultures of human fetal glial cells, producing infectious virus as early as 1 week after viral adsorption. Their brain-cell derivation has been established as astroglial, based on their reactivity with a monoclonal antibody to glial fibrillary acid protein and lack of activity with an anti-galactocerebroside antibody, which identifies oligodendroglial cells. The SVG cells represent a unique line of continuous rapidly growing human fetal astroglial cells that synthesizes a replication-proficient SV40 T protein. Their susceptibility to JC virus (JCV) infection obviates a host restriction barrier that limited JCV studies to primary cultures of human fetal brain and thus should allow for more detailed molecular studies of human brain cells and JCV that infects them.