Epileptic seizures are known to occur in different animal models of demyelination and have also been described in demyelinating diseases of the central nervous system (CNS) such as multiple sclerosis. How myelin deficiency might cause seizures is unknown, but may involve axonal pathology and resultant alterations in neuronal excitability. The cause of seizures occurring in rodent demyelination models is unknown. In the present study, we used EEG/video monitoring to record seizures occurring during chronic demyelination of C57BL/6 mice fed for 12 weeks with 0.2% cuprizone. Furthermore, in the search for a morphological correlate of the seizures, the hippocampal formation was examined histologically. Epileptiform spikes resembling interictal spikes known from chronic epilepsy were recorded in all cuprizone-treated mice, but not in controls. Most cuprizone-treated animals exhibited generalized tonic-clonic seizures upon stress-inducing stimuli. In addition to the known demyelination of the corpus callosum, massive demyelination was found in the hippocampal formation. This was associated with neuronal alterations, including a loss of neurons in the hilus of the dentate gyrus. In view of the role of the dentate gyrus in epileptogenesis, demyelination leading to axonal pathology and thus neuronal damage as observed in the hilus may be causally involved in the paroxysmal alterations observed after prolonged treatment with cuprizone. The present data suggest a potential role of the hippocampal formation for seizures occurring as a consequence of neuronal damage secondary to CNS demyelination.