The relationship between hippocampal activity and the extracellular level of excitatory amino acids in the median raphe nucleus has been studied in urethane anaesthetized rats, using the in vivo microdialysis technique. Dialysates were collected from the median raphe nucleus during two to eight sampling periods of equal length (20 min) and hippocampal electroencephalogram was continuously monitored. For each observation period, the average glutamate level in the median raphe nucleus was determined and the percentage of theta and non-theta segments in the hippocampal recordings was calculated. Theta synchronization, in these experiments, either developed spontaneously or it was elicited by injection of anticholinesterase (Physostigmine or Sintostigmine, i.p.) or by a series of short tail pinches. The relationship between hippocampal activity and glutamate release in the median raphe nucleus was characterized by comparison of the direction of changes in these two parameters in consecutive sampling periods. We found that as long as theta/non-theta ratio changed spontaneously or under the effect of anticholinesterase (n = 7), the extracellular level of glutamate in the median raphe nucleus was elevated during periods dominated by desynchronized hippocampal activity as compared with those mostly containing long and/or frequently occurring theta segments. Such relationship was not observed in the adjacent reticular formation (n = 4) and in the median raphe nucleus during sensory stimulation (n = 2). The present data complete those found earlier indicating that the desynchronizing serotonergic influence originating from the brainstem is maintained by a tonic excitatory input to the median raphe nucleus. Since the majority of glutamatergic afferents to the median raphe nucleus originates from the lateral habenula and the interpeduncular nucleus, known to connect limbic forebrain to the brainstem, theta associated changes in median raphe nucleus glutamate levels might reflect descending forebrain influences, suggesting therefore a feedback regulation of the hippocampal activity involving brainstem structures.