Damage to neurites with transection of axons and spheroid formation is commonly noted in the central nervous system during viral and autoimmune diseases such as multiple sclerosis, but it remains open whether such changes are caused primarily by immune mechanisms or whether they are secondary to inflammation. The present experiments explored whether neurites can be directly attacked by cytotoxic T lymphocytes (CTLs). Cultured murine neurons induced by interferon-gamma and tetrodotoxin to express major histocompatibility complex class I were pulsed with a dominant peptide of the lymphochoriomeningitis virus envelope glycoprotein (GP33) and then confronted with GP33-specific CD8(+) CTLs. Within 3 hours the neurites developed cytoskeleton breaks with adjacent solitary neuritic spheroids, as documented by confocal examination of the cytoskeletal marker beta-tubulin III. At the same time cytoskeleton staining of the neuronal somata showed no damage. The CTLs selectively attacked neurites and induced segmental membrane disruption 5 to 30 minutes after the establishment of peptide-specific CTL-neurite contact, as directly visualized by live confocal imaging. Thus, major histocompatibility complex class I/peptide-restricted CD8(+) T lymphocytes can induce lesions to neurites, which might be responsible for axonal damage during neuroinflammatory diseases.