To study the pathogenesis of demyelination in canine distemper virus (CDV) infection, primary canine brain cell cultures were infected with CDV to examine specific virus-induced glial cell changes. Cultures were harvested at regular intervals after inoculation and were immunostained for the specific demonstration of astrocytes, oligodendrocytes, and CDV antigen. The infection spread slowly with moderate cytolysis and cell fusion. Soon after inoculation, infection of astrocytes was found by means of double immunofluorescent labeling. One week after inoculation, CDV-induced astrocytic fusion and rearrangement of astroglial fibrils became apparent. The astrocytic changes progressed during the observation period. Double immunofluorescent labeling failed to show oligodendroglial infection. Despite clear absence of viral replication within oligodendrocytes at all stages of the experiment, these cells exhibited marked pathologic changes starting at about 20 days after inoculation and progressed to complete cytolysis within 10 days. The degeneration of the oligodendrocytes was thought to be secondary to CDV-induced changes in other cell types of the culture probably through the release of toxic factors in the tissue culture medium. Since little evidence has been found for oligodendroglial infection in demyelinating lesions in canine distemper in vivo, the present tissue culture findings suggest that demyelination in vivo could be the result of indirect oligodendroglial damage caused by CDV-induced changes in other cell types such as astrocytes.