The ultrastructural pathology of myelinated axons in mice infected experimentally with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus is characterized by myelin sheath vacuolation that closely resembles that induced in murine spinal cord organotypic cultures by tumor necrosis factor [Alpha] (TNF-[Alpha]), a cytokine produced by astrocytes and macrophages. To clarify the role of TNF-[Alpha] in experimental CJD, we investigated the expression of TNF-[Alpha] in brain tissues from CJD virus-infected mice at weekly intervals after inoculation by reverse transcription-coupled PCR, Northern and Western blot analyses, and immunocytochemical staining. Neuropathological findings by electron microscopy, as well as expression of interleukin 1[Alpha] and glial fibrillary acidic protein, were concurrently monitored. As determined by reverse transcription-coupled PCR, the expression of TNF-[Alpha], interleukin 1[Alpha], and glial fibrillary acidic protein was increased by approximately 200-fold in the brains of CJD virus-inoculated mice during the course of disease. By contrast, [Beta]-actin expression remained unchanged. Progressively increased expression of TNF-[Alpha] in CJD virus-infected brain tissues was verified by Northern and Western blot analyses, and astrocytes in areas with striking myelin sheath vacuolation were intensely stained with an antibody against murine TNF-[Alpha]. The collective findings of TNF-[Alpha] overexpression during the course of clinical disease suggest that TNF-[Alpha] may mediate the myelin sheath vacuolation observed in experimental CJD.