Brainstem serotonergic neurotransmission is implicated in sleep regulation. However, the role of serotonin (5-HT) in forebrain regions in sleep–wake mechanisms is still unclear. Here, we have investigated, using a combined in vivo microdialysis/electroencephalogram method, the relationship between hippocampal 5-HT levels and sleep–wake behaviour in the rat. A clear-cut relationship was found between hippocampal 5-HT levels and vigilance state. The highest levels of 5-HT were observed during wakefulness, whereas a progressive decrease of 5-HT going from nonrapid eye movement sleep to rapid eye movement sleep was found. Sleep deprivation (SD) causes a transient enhancement of mood in depressed patients. Given the putative role of 5-HT in the aetiology of depression and the therapeutical efficacy of selective serotonin reuptake inhibitors in this illness, we also studied hippocampal 5-HT during 4 h of SD and during the subsequent recovery period. During the whole SD period, 5-HT levels were elevated substantially when compared to 5-HT levels during basal wakefulness. However, no changes in 5-HT levels and the relationship between hippocampal 5-HT and vigilance state were found during the subsequent recovery period. As SD is a potentially stressful experience and glucocorticoids are involved in the regulation of serotonergic neurotransmission and sleep, we investigated the effects of SD on free corticosterone levels. SD caused a marked rise in free corticosterone levels. However, the effects of SD on 5-HT seem not to be mediated by this hormone, because adrenalectomy did not affect the rise in hippocampal 5-HT during SD. We hypothesize that the elevated hippocampal 5-HT levels during SD may participate in the transient mood enhancing properties of forced wakefulness observed in depressed patients.