The highly restricted host range of JC virus (JCV) has made it difficult to study the biology of this common human papovavirus. To increase our understanding of the tissue specificity of this virus, we have examined the expression of the T antigen (T-Ag) in primary and established cell lines from various tissues of transgenic mice containing the JCV early region. In contrast to earlier results from a simian virus 40-containing transgenic mouse, there was no T-Ag expression in mesenchymal fibroblasts derived from two lines of JCV-transgenic mice. Instead, we isolated T-Ag-positive (T-Ag+) cells that had characteristics consistent with a neural crest origin. Furthermore, primary brain cultures contained many T-Ag+ astrocytes, but no expression was detected in macrophages, epithelial cells, neuronal cells nor, surprisingly, in oligodendrocytes. Continued passage of these cultures resulted in vigorously growing glial fibrillary acidic protein-positive, T-Ag+ astrocytes. Thus, the strict tissue specificity of JCV expression was maintained, despite the fact that the viral genome pre-existed in every tissue of these transgenic mice and these constraints on expression were preserved even when cells were explanted in vitro.