The hippocampus is processing temporal and spatial information in particular contexts or episodes. Using freely moving rats, we monitored extracellular levels of acetylcholine (ACh), a critical neurotransmitter activating hippocampal circuits. We found that the ACh release in the dorsal hippocampus increases during the period of learning or exploration, exhibiting a sex-specific 24-h release profile. Moreover, neonatal increase in circulating androgen not only androgenizes behavioral and hormonal features, but also produces male-type ACh release profile after the development. The results suggest neonatal sexual differentiation of septo-hippocampal cholinergic system. Environmental conditions (such as stress, housing or food) of animals further affected the ACh release.Although recent advances of neuroscience successfully revealed molecular/cellular mechanism of learning and memory, most research were performed using male animals at specific time period. Sex-specific or time-dependent hippocampal functions are still largely unknown.