Previous findings from our laboratory have demonstrated a positive correlation between the development of tolerance to diazepam (DZ) 5 mg/kg/day over 4 days, and increased hippocampal synaptic plasticity. It seems likely that a similar plastic phenomenon may occur on hippocampal formation after chronic (18 days) DZ administration. We postulate hippocampal long-term potentiation (LTP) underlying substrate to the behavioral alteration observed after chronic DZ administration. In the present study, we investigated the involvement of the serotonergic (5-HT) system in the possible neural circuitry recruited during DZ withdrawal and in the increased hippocampal synaptic plasticity associated with the discontinuation of chronic DZ administration. The results of the current research demonstrate an increased neuronal activity in the dorsal raphe nucleus (DRN) during withdrawal. Previous MK-801 administration impairs the development of anxiety signs observed during withdrawal and the concomitant increased electrical activity on 5-HT neurons on DRN. These results are discussed in terms of the participation of 5-HT system in the modulation of hippocampal plasticity developed on DZ withdrawal. Synapse 56:61–68, 2005. © 2005 Wiley-Liss, Inc.