The observation of murine retrovirus infection of microglial cells in brain regions expressing spongiform neurodegenerative changes suggests that these cells may play an important role in pathogenesis. To evaluate this potential in vitro, murine microglial cells were infected in mixed glial cultures with the highly neurovirulent murine retrovirus, FrCasE. The microglia were then isolated from the mixed cultures on the basis of their differential adherence and shown to be approximately 98% pure. The infected microglia expressed viral envelope protein at the plasma membrane, while viral budding was primarily intracellular. Evaluation of the viral envelope protein by immunoblotting indicated that the immunoreactive species produced was exclusively a 90-kDa precursor protein. Very little of the envelope protein was associated with particles released from these cells, and viral titers in the culture supernatant were low. Interestingly, these cells were still capable of infecting permissive target cells when seeded as infectious centers. This partially defective infection of microglial cells suggests a potential cellular means by which a neurovirulent retrovirus could disrupt normal microglia and in turn central nervous system motor system functioning.