Cyclothiazide (CTZ) is a potent blocker of AMPA receptor desensitization. We have recently demonstrated that CTZ also inhibits GABAA receptors (Deng & Chen, 2003). Here we report that CTZ induces robust epileptiform activity in hippocampal neurons both in vitro and in vivo. We first found that chronic treatment of hippocampal cultures with CTZ (5 µM, 48 hours) results in epileptiform activity in the majority of neurons (80%). The epileptiform activity lasts more than 48 hours after washing off CTZ, suggesting a permanent change of the neural network properties after CTZ-treatment. We then demonstrated in in vivo recordings that injection of CTZ (5 µmol in 5 µL) into the lateral ventricles of anesthetized rats also induces spontaneous epileptiform activity in hippocampal CA1 region. The epileptogenic effect of CTZ is likely due to its enhancing glutamatergic neurotransmission as shown by increasing the frequency and decay time of mEPSCs, and simultaneously inhibiting GABAergic neurotransmission by reducing the frequency of mIPSCs. Comparing to a well-known epileptogenic agent kainic acid (KA), CTZ affects neuronal activity mainly through modulating synaptic transmission without significant change of the intrinsic membrane excitability. Unlike KA, which induces significant cell death in hippocampal cultures, CTZ treatment does not result in any apparent neuronal death. Therefore, CTZ-induced epilepsy model may provide a novel research tool to elucidate the molecular and cellular mechanisms of epileptogenesis without any complication from drug-induced cell death. The long-lasting epileptiform activity after CTZ washout may also make it a very useful model in screening antiepileptic drugs.