Abstract: Melatonin serves as a signal of darkness and participates in sleep/wake regulation. Animal studies demonstrated effects of melatonin in the hippocampus, particularly suggesting involvement in synaptic plasticity. We used functional magnetic resonance imaging to identify and investigate effects of melatonin in the human hippocampus. Activity in the hippocampal complex during a memory task was examined at 22:00 hr (when endogenous melatonin levels are normally increasing) and compared with 16:00 hr (when endogenous melatonin levels are minimal). The relationship between observed activation patterns and endogenous melatonin was assessed. Finally, the effects of exogenous melatonin administered at 22:00 hr were studied in a double-blind, placebo-controlled crossover manner. Our findings indicate that activation in the left hippocampus at 22:00 hr is significantly reduced compared with afternoon hours compatible with diurnal variation in hippocampal activity. Exogenous melatonin further reduced activation in this region, only in subjects who already crossed the melatonin onset phase at this hour and in correlation with endogenous melatonin levels. As such an effect was not demonstrated with afternoon administration of melatonin, a time depended effect is suggested. Contrary, activation in the left parahippocampus at 22:00 hr was higher in subjects that crossed the melatonin onset phase. Parahippocampal activation correlated with individual endogenous melatonin levels and was not further affected by exogenous melatonin. These results demonstrate that memory related activation in the hippocampus and parahippocampus are affected by time of day and melatonin in a differential manner and may implicate the circadian clock and melatonin in human memory processing during the night.