Primary dissociated ovine brain cell cultures prepared from 50- or 140-day-old fetuses were inoculated with border disease virus (BDV). The cells present in the cultures were identified, using immunofluorescence procedures and sera against various CNS cell-specific markers. These markers were glial fibrillary acidic protein, myelin basic protein, myelin-associated glycoprotein, neuron-specific enolase, neurofilament protein, and fibronectin. Double-labeling immunofluorescence techniques for visualization of BDV antigen and of the CNS cell markers were used to evaluate the pattern of individual cell susceptibility 48 hours after infection. In cultures from fetuses of both ages, about half of the infected cells were glial fibrillary acidic protein-positive astrocytes. Scattered myelin-associated glycoprotein-positive oligodendrocytes were positive for BDV antigen, but only in the infected cultures from the older fetuses. Fibronectin-positive cells were not infected with BDV. In infected and noninfected cultures, cells positive for neuron-specific enolase, myelin basic protein, or neurofilament protein were not seen. Therefore, the remaining infected cells in all the cultures were not identified by the cell-specific markers used. Results of these in vitro experiments indicate that BDV does not selectively infect oligodendrocytes, and that such a selective pattern of infection may not be the basis for the in vivo congenital hypomyelination in sheep with border disease.