Demyelination and death of oligodendrocytes accompanied by transection of neurites and neuronal apoptosis are pathological hallmarks of cortical and subcortical gray matter lesions in demyelinating viral and autoimmune inflammatory CNS disorders. In these disorders, leukocortical lesions, containing the perikarya of most efferent neurons, display pronounced infiltration by CD8⁺ T cells of putative specificity for oligodendrocyte‐ and myelin‐related antigens. Hence, neuronal apoptosis in gray matter lesions may be a collateral effect of an oligodendrocyte‐directed attack by CD8⁺ T cells. To challenge this hypothesis, we transferred activated antigen‐specific CD8⁺ T cells (OT‐I T cells) into acute coronal brain slices from mice selectively expressing ovalbumin as a cytosolic neo‐self‐antigen in oligodendrocytes (ODC‐OVA mice). We studied mechanisms and kinetics of oligodendroglial and neuronal apoptosis in the neocortex and hippocampus, using multicolor staining for different cell types and activated caspase‐3. Within the gray matter, a single OT‐I T cell caused simultaneous caspase‐3 activation in about 30 ODCs and 10 neurons within 6 h in a strictly antigen‐dependent manner. Experiments with OT‐I T cells genetically deficient for perforin or the granzyme B‐cluster and with blocking anti‐FasL antibodies as well as proinflammatory cytokines revealed, that collateral apoptosis of neurons was likely due to a spillover of perforin and granzyme(s) from the OT‐I T cell itself or the immunological synapse that it selectively formed with antigen‐presenting oligodendrocytes. Collateral neuronal apoptosis could contribute to substantial neuronal loss in gray matter lesions and cause persistent neurological impairment in both acute and chronic gray matter lesions in various inflammatory CNS disorders. © 2009 Wiley‐Liss, Inc.